# encyclopedia

details:
- There are 2 types:

- N-glycosylation:
   - occurs in E.R.
   - Important for folding of some eukaryotic proteins
   - Occurs by addition of sugar chains at the amide 'N' Nitrogen of the side chain of amino acid Asparagine [AAT, AAC].

- O-glycosylation:
   - occurs at later stage in the golgi app.
   - occurs by addition of sugar chains at the hydroxyl 'O' oxygen of the side chain of hydroxy-lysine, or hydroxy-proline, or serine, or therionine.


details:
When do I need to use a logarithm 'log' to represent my data on a graph?

- When the numbers you want to represent on one of the axes of a graph lie in different scales, [e.g, 2.00, 6.00, 50.0, 6.00 x 10^3, 2.20 x 10^5, 1.65 x 10^7 representing a cell counts in different time points].

- OR to easily represent data of large values.

- Log 'logarithm'is the power [or exponent] to which the (base) must be raised in order to reach to the number.

- e.g, log 1000 to base (10) = 3, meaning that the base (10) must be raised to power 3 [i.e, multiplied 3 times] to get 1000.

- For bigger  numbers, you can use a calculator to get the log, it is also available in different web sites.

- then, make your table and graph and indicate the log (base) on the relevant axis e.g, log(10).

*Image 2:  x = time (hours) & Y= cell count [log(10)]


details:
Hazard: the ability of a substance or biological agent to cause Harm.


Risk: the probability of a substance or biological agent to cause harm in a specific situation.


How to differentiate between Hazard and Risk: 

One of the Hazards associated with water is drowning. 
However, the Risk of drowning in a few drops of water is minimal !!


details:
Hazard groups: [according to UK Advisory Committee on Dangerous Pathogens (ACDP)]

1 	Unlikely to cause human disease.
2 	May cause disease
   Possible hazard to laboratory workers / minimal hazard to community.
3 	May cause sever disease
   May be a serious hazard to laboratory workers / may spread to community.
4 	Causes sever disease
   Is a serious hazard to laboratory workers / high risk to community.


details:
4 bio-safety levels (BSLs) according to different laboratory techniques, safety equipment, and design, depending on the types of agents being studied: 

BSL-1 labs: used to study agents not known to consistently cause disease in healthy adults. 
       - Basic safety procedures - no special equipment or design features. 

BSL-2 labs: used to study moderate-risk agents that pose a danger if accidentally inhaled, swallowed or exposed to the skin. 
       - Safety measures: gloves - eyewear - hand washing sinks - waste decontamination
         facilities. 

BSL-3 labs: used to study agents that can be transmitted through the air and cause potentially lethal infection.  
       - Safety measures: Gas-tight enclosure - specialized ventilation system - clothing 
        decontamination - sealed windows. 

BSL-4 labs: used to study agents that pose a high risk of life-threatening disease for which no vaccine or therapy is available. 
      - Safety measures: wearing full-body, air-supplied suits - shower when exiting the facility - occupy safe, isolated zone within the building.


details:
Incidence: 
number of new cases of a disease in a specified period of time

Incidence rate: 
number of new cases of a disease per 'unit of person-time' at risk.

Prevalence: 
total number of cases of a disease in a specified period of time


details:
Cryo-EM overcomes the disadvantages of the EM in which 'heavy metals' are used for staining the biological material to be stained causing many artefacts such as shrinkage, distortion, and flattening of particles, however, in cryo-EM the biological material is suspended in 'vitreous ice' which is obtained by 'extremely rapid' cooling of water to -140 to avoid formation of "ice crystals", however it forms 'amorphous ice' with glass-like quality.

Stains used with the EM: 
- Silver nitarate
- Phosphotungestic acid


details:
Class I DS DNA

Class II SS DNA

Class III DS RNA

Class IV SS RNA + sense

Class V SS RNA - sense

Class VI SS RNA, RT viruses + sense with DNA intermediate 'Retroviruses'

Class VII DS DNA, RT viruses 'Hepadnaviruses'


details:
family --> genera --> genogroups --> genotypes 'genetic clusters' --> strains 'genetic variants'


details:
Many general purpose vectors such as pUC19 usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the gene coding for E. coli β-galactosidase, whose integrity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal (5 bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo). Cloning a fragment of DNA within the vector-based gene encoding the β-galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.


details:
Description: 
Pfu DNA Polymerase(a) is a thermostable enzyme that replicates DNA at 75°C. It catalyzes the polymerization of nucleotides into duplex DNA in the 5´→3´ direction in the presence of magnesium. The enzyme has a molecular weight of approximately 90,000 daltons as estimated from the predicted amino acid sequence and exhibits 3´→5´ exonuclease
(proofreading) activity. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require
high fidelity.

Extension Time:
The extension rate of Pfu DNA Polymerase is lower than that of Taq DNA Polymerase.
Therefore, during the extension step, allow approximately 2 minutes for every 1kb to be amplified (minimum extension time of 1 minute). For most reactions, 25–35 cycles are sufficient.

Note: It is critical to withhold Pfu DNA Polymerase until after the addition of dNTPs; otherwise, the proofreading activity of the polymerase may degrade the primers, resulting in nonspecific amplification and reduced product yield. Assemble on ice.

Amount:
Pfu DNA Polymerase (2–3u/μl) ---> use  1.25u/50μl and 
2.5u/100ul reaction [ie: 1ul]

Recommended thermal cycling conditions for Pfu DNA Polymerase-mediated PCR amplification. 
These guidelines apply to target sequences between 200 and 2,000bp and are optimal for the Perkin-Elmer Thermal Cycler Model 480 or comparable thermal cyclers.
Step               Temperature              Time                Number of Cycles
Initial Denaturation 95°C              1–2 minutes                   1 cycle
Denaturation          95°C              0.5–1 minute
Annealing*          42–65°C             30 seconds              25–35 cycles
Extension**         72–74°C             2–4 minutes
Final Extension    72–74°C              5 minutes                   1 cycle
Soak                      4°C                    Indefinite                   1 cycle

*The annealing temperature for a specific amplification reaction will depend upon the sequences of
the two primers. See Section III for discussions on determining optimal annealing temperatures for
PCR amplification.


details:
Description:
T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or
blunt-ended configuration (1). The enzyme has also been shown to catalyze the joining of RNA to either a DNA or RNA strand in a duplex molecule but will not join singlestranded nucleic acids.

optimum temperature:

There is considerable latitude in the temperature and time needed for successful ligations. The optimal temperature for a ligation is a balance between the optimal temperature for T4 DNA Ligase enzyme activity (25°C) (1) and the temperature
necessary to ensure annealing of the fragment ends, which can vary with the length and base composition of the overhangs. Shorter duplexes (linkers less than 16 bases
long) require lower temperatures as a result of their lower melting temperatures (Tm). In general, ligation reactions performed at lower temperatures require longer
incubation times. The scientific literature reflects this variability in ligation conditions. Blunt-end ligations generally are efficient at temperatures between 15–20°C for 4–18 hours, while sticky ends are ligated effectively at room temperature (22°C) for 3 hours or 4–8°C overnight.


details:
A cloning vector is a small DNA vehicle into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Most commercial cloning vectors have key features that have made their use in molecular biology so widespread.

In the case of expression vectors, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (eg. E. coli). Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower mosaic virus's 35s promoter (for plant vectors).

Most commercial cloning vectors have key features that have made their use in molecular biology so widespread. 

In the case of expression vectors, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (eg. E. coli). Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower mosaic virus's 35s promoter (for plant vectors).


To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a synthetic multiple cloning site (MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic resistance [eg. beta-lactamase] is often carried by the vector to allow the selection of positively transformed cells. All plasmids must carry a functional origin of replication.

Some other possible features present in cloning vectors are: vir genes for plant transformation, intergrase sites for chromosomal insertion, lacZα fragment for α complementation and blue-white selection, and/or reporter genes in frame with and flanking the MCS to facilitate the production of recombinant proteins [eg. Fused to the Green fluorescent protein (GFP) or to the glutathione S-transferase].


title:
RELATIVE CONTRIBUTIONS OF HOST DEFENSE MECHANISMS

details:
in virus infection:

"The relative contribution of the various host defense mechanisms will depend on the nature of the virus and the portal of entry. Antibodies will be more important in infections in which viremia is a prominent feature. However, antibodies may not be helpful in infections with herpes or paramyxoviruses in which the virus can be passed from cell to cell by cell fusion. In this instance cell mediated immunity is more important. If a virus only infects cells in the mucosal surface, IgA antibodies may be important.

An understanding of the host defense mechanisms is important for vaccine development and for proper administration of vaccines. If IgA antibodies are important for protection against a particular virus, then any vaccine must be able to stimulate production of IgA antibodies in the appropriate mucosal surface. Alternatively if CTLs are important then the vaccine must be able to stimulate CTL production. That is why live vaccines are often preferable to a killed vaccine because live vaccines usually lead to the generation of CTLs while killed vaccines do not".

Ref. microbiology and immunology , univ of south carolina


details:
- study of evolutionary relatedness among various groups of organisms  

- is done by relating the molecular sequencing data and morphological data

-  Gene transfer includes: vertical gene transfer and horizontal gene transfer. 

- Vertical gene transfer: is the passage of genes from parent to offspring, 

- horizontal gene transfer: (lateral gene transfer) occurs when genes jump between unrelated organisms, a common phenomenon in prokaryotes.

- Horizontal gene transfer has complicated the determination of phylogenies of organisms as it causes inconsistencies in phylogeny which have been reported among specific groups of organisms depending on the genes used to construct evolutionary trees.


details:
is the Freeze-drying 
by freezing the material and then reducing the surrounding pressure and adding enough heat to allow the frozen water in the material to sublime directly from the solid phase to gas.


details:
number of persons in a population who test positive for a specific disease based on serology


details:
M13 forward sequencing primer (-20): GTAAAACGACGGCCAGT 
M13 forward sequencing primer (-40): GTTTTCCCAGTCACGAC 
M13 forward sequencing primer (-47): CGCCAGGGTTTTCCCAGTCACGAC 
M13 reverse sequencing primer: (-24): AACAGCTATGACCATG 
M13 reverse sequencing primer: (-48): AGCGGATAACAATTTCACACAGGA


details:
- Number of Sf21cells at 70% confluency which is the ideal for doing infection with Baculovirus:

- Medium-sized flask [75 cm^2] = ~ 1.6 x 10^7
- Small-sized flask [25 cm^2] = ~ 5 x 10^6
- 35mm dish [3.5 cm^2] = ~ 2 x 10^6
- Roller bottle [850 cm^2]= 12 times as the medium-sized flask = 12 x 1.6 x 10^7 = 2 x 10^8

* for viral plaque assay, the cells should be ~50% confluent

- Amount of growth medium required:
  - Medium-sized flask = 15ml
  - Small-sized flask = 6ml

- Passage level from flasks (70% confluent) is 1:4 to 1:6, cells take ~ 3-4 days to reach 70% confluency
- Passage level from 35mm dish (70% confluent) is 1:16
- Passage should be done twice/week


details:
- Primers are short, single-stranded oligonucleotides designed to be able to anneal to template DNA and thus act as starting points for polymerase enzyme to amplify certain fragment of the template.

- There must be 2 primers in order to amplify a double-stranded DNA segment

- Each primer must be complementary to 1 strand of the DNA duplex

Primer design:
General considerations:
1.  primers should be 17-28 bases in length;   
2.  base composition should be 50-60% (G+C);   
3.  primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;   
4.  Tms between 55-80oC are preferred;   
5.  3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;   
6.  primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided;   
7.  runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.   
(adapted from Innis and Gelfand,1991).

………………………………………………

- Forward primers are similar to the top strand of DNA ie. The one appearing in the example.
- Reverse primers are similar to the bottom strand of DNA ie. The complementary to the one appearing in the example. 
- Be careful when writing the reverse primers for ordering (it must be the complement to the 5’-3’ strand and read from right to left while writing it from left to right). 
- Usually add 2-3 extra bases outside the restriction enzyme site which give space for the enzyme to attach and identify the site, so the final primer will be

- These extra bases (outside the restriction sites) do not matter as they will be removed in the restriction digestion reaction.
- However, the extra bases added beside the restriction sites towards the wild sequence will differ during translation of the protein either introduce stop codon, adjust the open reading frame, or move the frame to change some amino acids eg. Those added in the restriction site if we want to avoid certain amino acids, or add new amino acids to the wild protein.


Synthetic polyglycine nucleotide chains:
	
Name	Nucleotide sequence
Annealed    8 poly-glycine
‘Sense’ & ‘Antisense’   strands	
5’-TCG AGT GGA GGA GGA GGA GGA GGA GGA GGA 3’
3’-           CA CCT  CCT  CCT  CCT  CCT  CCT  CCT  CCTTCGA-5’


details:
The bacterial transposon Tn7 is an unusual mobile DNA segment. Most transposable elements move at low-frequency and display little target site-selectivity. By contrast, Tn7 inserts at high-frequency into a single specific site in the chromosomes of many bacteria. In the absence of this specific site, called attTn7 in Escherichia coli where Tn7 has been most extensively studied, Tn7 transposes at low-frequency and inserts into many different sites. Much has recently been learned about Tn7 transposition from both genetic and biochemical studies. The Tn7 recombination machinery is elaborate and includes a large number of Tn7-encoded proteins, probably host-encoded proteins and also rather large cis-acting transposition sequences at the transposon termini and at the target site. Dissection of the Tn7 transposition mechanism has revealed that the DNA strand breakage and joining reactions that underlie the translocation of Tn7 have several unusual features.Tn7


details:
Ionic bonding is a type of chemical bond that involves the electrostatic attraction between oppositely charged ions. These ions represent atoms that have lost one or more electrons (known as cations) and atoms that have gained one or more electrons (known as an anion).


title:
how a protein acts as a primer for transcription

details:
VPg (viral protein genome-linked) is a protein that is covalently attached to the 5′ end of positive strand viral RNA and acts as a primer during RNA synthesis in a variety of virus families including Picornaviridae and Caliciviridae. The primer activity of VPg occurs when the protein becomes uridylylated, providing a free hydroxyl that can be extended by the virally encoded RNA-dependent RNA polymerase. Aside from functioning as a primer, VPg also has a role in translation initiation by acting like a 5' mRNA cap.

VPg was first described in initial investigations of poliovirus RNA as a protein covalently attached to the 5' end of the genome.[1][2] and later seen in caliciviruses.[


# machine details

maintenance company:
Philip Harris limited shenstone - England


# machine name

machine name:
cytogenetics  and FISH fluorescent microscope


# mechanism of action

mechanism of action:
stabilizes the enzymes, prevent adhesion of the enzymes to the wall of the reaction tube


technique:
Digestion of DNA using restriction enzymes


mechanism of action:
catch the non-specific antibodies to prevent the flase positive results


mechanism of action:
- Chelate metal ions so, deactivates metal-dependant enzymes.

- preventing Dnases from damaging the plasmid

- helps in destabilizing the cell wall.


mechanism of action:
1- Mg complexes with the single nucleotides 'dNTPs', Only Mg/nucleotide compleses are the substrate for the DNA polymerase.

2- Mg promotes DNA/DNA interaction
so, if too low --> primer fails to anneal to the template DNA
if too high --> base pairing becomes so strong and the amplicon fails to denature completely at 94c


mechanism of action:
- helps to break down the cell wall

- it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and plasmid, to single stranded DNA (ssDNA). This process is called denaturation


mechanism of action:
Glycerol is much more dense than water and is added to make the sample fall to the bottom of the sample well rather than just flow out and mix with all the buffer in the upper reservoir.


mechanism of action:
ionic detergent that binds to the vast majority of proteins at a constant ratio of 1.4 gm SDS/gm protein. A few proteins like tubulin do not bind at this ratio and this is one reason why some proteins migrate anomalously (there are other reasons as well so you shouldn’t put too much faith in the apparent molecular weight estimated from an SDS PAGE gel).  Since SDS is an anionic detergent it imparts a negative charge to all the proteins in your sample. More importantly, these charges swamp the inherent charge of the proteins and give every protein the same charge-to-mass ratio. Because the proteins have the same charge-to-mass ratio, and because the gels have sieving properties, mobility becomes a function of molecular weight


mechanism of action:
ethanol will give a flat surface. 
Also, since polymerization of acrylimide is inhibited by oxygen it will speed up polymerization.


# methods

steps:
- 1.2% agarose is used to resolve DNA samples according to their size. 
- prepare 1.2% agarose, pour in the cast, put the comb tell the gel sets and remove the comb.

- 10µl of each DNA sample is mixed with 10µl 2x TBE sample buffer, and loaded into the gel, 

- 10µl of 1kb DNA ladder 'marker' is also loaded. 

- Electrophoresis proceeds at 125 volts for about 1 hour using TBE running buffer. 

- DNA bands are visualised using a UV trans-illuminator and images are digitally recorded using UV machine.


steps:
- PCR uses:
1- amplify target DNA sequences
2- screen transformants and recombinants for existance of the correct insert
3- mutagenesis. 

- A 100µl PCR reaction typically contains
 10µl 10x PCR buffer, 
 10µl 2mM dNTPs mixture, 
 10µl 2.5µM sense primer, 
 10µl 2.5µM anti-sense primer, 
 58µl MQ water, 
 2µl of the target DNA. 
 5 units of Taq polymerase enzyme 
Mix, buzz down
overlaid with 100µl liquid paraffin in reaction tubes and placed in a thermal cycler. 

- The machine is adjusted according to the amplified target DNA size and the melting temperatures 'Tm' of the primers, 

- as a rough guide extension times are calculated at 1minute/1000 bases (for Taq polymerase) and annealing temperature 'Ta' is 5-10c below the 'Tm' of the oligo primers used.


steps:
- cells should be thawed rapidly by holding the cryovial in water bath at 37c

- transfer the cells to a sterile universal tube

- add 5ml of the growth medium (TC-100), mix gently

- spin at low speed [2500 rpm] for 5 mins

- discard the supernatent

- resuspend the pellet in 5ml of growth medium

- distribute to flasks acc to the size of the pellet

- incubate at 28c overnight

- next day, change the medium to remove any residual DMSO 'dimethyl sulphoxide'

- re-incubate till 70% confluent i.e: 3 days


steps:
- warm the growth medium to room remp

- use non-vented flasks of suitable size

- check the confluency of cells

- pour off the old medium

- add 5ml fresh growth medium, use sterile glass beads to detach the cells by rolling the beads gently over the flask surface

- adjust the growth medium volume in the new flasks

- divide the cells between the new flasks [1:4 or 1:6]

- cells should be confluent again in 3-4 days

- passage twice/week


steps:
after the insertion of the gene of interest in baculovirus genome by transfection of the insect cells using the Lac z baculovirus linearized DNA and the shuttle vector 'pVL1393' containing the insert.

- use 5 large [175 cm2] non-vented flasks to grow lage no of sf21 cells

- when cells become confluent 70%, discard the old medium

- calculate the required volume of the viral inoculum acc to the required m.o.i
for m.o.i = 5 pfu/cell, use 3.3ml of virus inoculum [titre: 6 x 10 (7) pfu/ml]

- add 4ml of fresh growth medium

- rock the flasks at room temp. for 1 hour

- discard the inoculum

- wash the cells with PBS (or SF 900 serum free medium) 3 times using ~ 4ml/flask each time

- add 45ml sf 900 medium to each flask

- incubate flasks at 28c for 4-5 days till cpe is noticed

precautions:
- Antibiotics should be added to the sf900 medium to prevent contamination
penicillin/streptomycin  1%  i.e: 10ul /ml
and gentamicin  2.5 - 5 % i.e: 25-50 ul / ml
so, for 500ml medium add: 5ml P/S and 12.5ml G

im1lab:
purification steps of target protein using CsCl density gradient


steps:
1- Coating the plate with the antigens:

- Antigens are sonicated on ice [avoid the effect of heat] to dissocaite lumps

- Dilute antigens as required and according to the final concentration of your antigens e.g, I have ~20mg/ml, I diluted it to 10mg/ml and then made the following concentrations:
 e.g, 1.0, 0.5, 0.1, 0.01 mg/ml

- in the 96 well ELISA plate [12x8] image 1, add 100ul of each concentration starting from well number 1 and going 1,2,3,4 [4 wells].

- Repeat the same for each concentration according to the number of serum dilutions you intend to use e.g, 3 dilutions [1:200, 1:500, and 1:1000] so, repeat loading the antigens in A,B,C.

- ELISA must be done in triplicate or at least duplicates, so, you can repeat the same set in the same plate if you have enogh space with good markation of the wells.

- Also, a negative and positive controls must be included and in case of ELISA they are done using exactly the same dilutions as the sample antigen and the same sera.

- If the target antigen to be loaded [for the purpose of detection of antibodies against it in the produced sera] was produced in an expression system such as Sf21 cells or yeast cells, the negativeshould include the same cell proteins without the heterologous antigen [which is produced through transfection in case of Sf21 cells or trasformation in yeast with growing the cells for production]

- The positive control is a sample containing a confirmed specific antigen as the one against which we try to detect antibodies

- After adding the antigens to the wells the plates are incubated at 4 d.c overnight, and then, at 37 d.c for 2 hours

- Wash the wells once with phosphate buffered saline 'PBS' pH =7.4 +0.05% Tween-20 using a wash bottle

- Drain the wells by jerky movement in the sink and then on a tissue

- Dry at 37 d.c ---> you can keep the plates for future use at 4 d.c 'cold room' in a dissecator to avoid dryness

- Block the wells with gelatinous blocking solution [there are different blockng agents] for 2 hours at 37 d.c

- Drain the plates as before

- Test sera 'dilutions' are added 100ul to each well, incubated at 37 d.c for 1 hour, dilutions are made in PBS+ 1% bovine srum albumin 'BSA'

- wash 3 times with PBS-Tween-20, and drain as before

- Add 100ul of the suitable dilution of the suitable 2ry antibody-conjugate  [anti-globulin] e.g, 1:4000 [best know by asking the end-user] in PBS+ 1% bovine srum albumin 'BSA'
e.g, anti-mouse IgG/ HRP 'horse raddish peroxidase', incubate at 37 d.c for 1 hour

- Wash 3 times with PBS-T-20, and drain as before

- Add 100ul of the substrate 'TMB for HRP' to each well and incubate at room temperature for 15 minutes

- If the developing colour is not so intense and developing slowely you can read immediately after the incubation time at 650 nm in ELISA reader or if the colour is developing rapidly, use a stop solution 0.5M HCl for increasing the sensitivity and then read at 450 nm 

- Transfer your result to excel sheet and analyse by calculating the average of the triplicate or duplicate sets of the of the sample and the controls 

- Calculate the background by subtracting the values of the negative control from the sample to adjust the dilution which give the best results.


Technique:
Digestion of DNA using restriction enzymes

steps:
- One of its uses is to screen the results of ligations after transformation and plasmid isolation

- Another use is to cut a certain piece of DNA to be extracted from agarose gel and ligated in another plasmid vector e.g, expression vector or shuttle vector for homologous recombination of that piece into the genome of an expression host

- also, is used to linearise a recombinant plasmid fir transformation.

- Select the most efficient enzyme reaction buffer which allows the highest percentage of cutting from the provided manual such as promega enzymes

- thaw enzyme buffer and BSA ‘bovine serum albumin’ 

- prepare the digestion reaction 'restriction reaction' mix: 10ul
                 1ul      10x enzyme buffer         
                 1ul      10x BSA                         
                 3ul       DNA plasmid                
[or whatever acc. To the amount required to be cut either for just screening or for another cloning]
                 5ul       water                              
[or up to 10ul] acc to the volume of DNA sample

- Mix
- add restriction enzymes 1ul each [if they cut properly in the same buffer]

- mix with vortex, spin down the mixture

- incubate at 37 d.c in heat block for at least 1hour [1-2hs acc. to the amount of DNA to be cut]  
 
- for electrophoresis: add 2x sample buffer in the same volume [to be 1x] ie 10ul 2x loading buffer, load the whole amount 20ul into 1.2% agarose, run, use marker as 1Kb 10ul

* cutting large amount of DNA e.g. 2ug for gel extraction, use 45ul reaction

5ul 10x buffer
5ul 10x BSA
2ul DNA
34ul water
mix, add 2ul each enzme, 2 hours
- for electrophoresis:
add 6ul 10x TBE
15ul Glyycerol & dyes
run in 3 lanes, gel extract the DNA

precautions:
[check if both enzymes cut properly in the same buffer, and at the same temp.,otherwise
  you need to do cutting on 2 steps with DNA clening in between]


steps:
- use 6 well (3.5 cm2) tissue culture plates.

- seed with Sf21 cells in 2 ml growth medium/well. 

- incubate overnight at 28ºC or to 50% confluence. 

- Baculovirus stock to be assayed is serially diluted (10 fold) in serum-free TC100 medium.

- discard old growth medium and add 0.2ml of each dilution per well. 

- rock plates for 1 hour at room temperature, before the inoculum is removed and the cell sheets overlaid with 2 ml 1% seaplaque agarose in TC100 growth medium. 

- When the agarose is set, add 1 ml of growth medium to the surface. 

- incubate plates at 28ºC in a humidified box for 5 days. 

- Plaques are stained by adding 1ml of 1:10 neutral red stock (0.33% w/v) in growth medium to the 1ml of the medium already present in each well. 

- Incubation is continued for 4 hours when the stain was removed and incubation continued for a further 1 hour to allow the stain to clear. 

- Neutral red is a vital stain and does not inactivate the viruses. 

- Thus it can be used to obtain viral clones by plating recombinant virus following transfections and selecting individual plaques for subsequent culture.


steps:
- is done to concentrate the DNA sample

- DNA is precipitated from PCR reaction mixture after removal of paraffin, if the PCR tubes are frozen, then removal of paraffin is easier as it thaws before the mixture.

- add 2 volumes [of the mixture] absolute ethanol 
  and 1/30th volume sodium acetate solution (3M, pH 5.0). 

- Allowed to stand at -20°C overnight (or for 2 hours at -80°C) before the DNA is collected by spinning at 13,000 rpm for 10 minutes in a microfuge. 

- The supernatant is carefully removed and the pellet 'very small' is washed twice with 70% ethanol, dried at 37°C in heat blook for 3 minutes 
- resuspended in 20µl MQ water.
- vortex well for mixing


steps:
- Reactions usually include 20ng of the appropriate vector and sufficient insert DNA to achieve a 3:1 molar excess of insert to vector. 

- A 10µl ligation reaction includes
    1µl 10x ligase buffer 
    1µl 25% polyethylene glycol
    20ng of the vector
    and the calculated volume of the insert. 
    MQwater is added to bring the total volume to 10µl. 

- The reaction components are mixed and allowed to stand for 2 minutes on ice. 

- Then, 1µl of T4 DNA ligase is added and mixed. 

- The mixture is overlaid with 30µl of liquid paraffin and incubated overnight at 14°C in a water bath placed in a 4oC room. 

- After this time the ligation mix is diluted and the enzyme detached by addition of 1µl 0.2 M EDTA and 9µl Mill-Q water. 

- Ligation mixtures are stored at -20°C until they are used in transformation.


Technique:
Transformation of competent E.coli DH5α cells

steps:
- Ligated vectors are introduced into pre-prepared frozen competent E. coli cells strain ‘DH5α’. 

- Bacterial cell suspensions are thawed on ice and 50µl aliquots were transferred to pre-cooled 1.5ml Eppendorf tubes. 

- 10µl of diluted ligation mixture was added per tube and the E. coli cells, mixed gently and left to stand on ice for 30 minutes. 

- The transformation mixture is then heat shocked at 40ºC for exactly 45 seconds and returned to ice for 5 minutes. 

- 200µl of 2x YT 'Yeast extract Tryptone' medium was added and mixed. 

- Cells are then incubated at 37ºC in heating block for 1 hour with gentle mixing every 15 minutes to allow phenotypic expression. 

- The transformation mixture is then divided between (2x YT) agar pates containing appropriate selection antibiotic (usually ampicillin ‘200µl/100ml’), X-gal ‘5-bromo 4-chloro 3-indolyl β-galactosidase’ (500µl/100ml) and IPTG ‘isopropyl β- D-thiogalactopyranoside’ (250µl/100ml). 

- The later two components permit blue/white screening of recombinant colonies. 

- Four plates are used per tube of transformation mix. Plates are allowed to dry at room temperature and incubated in a 37ºC incubator overnight.


steps:
- The well separated 'white' colonies are picked from the transformation plates and grown separately overnight in 5ml 2x YT medium with vigorous shaking at 37ºC in an orbital shaker incubator. 

- Samples of each culture are stored separately at 4ºC whilst the bulk of the culture is sedimented in a microfuge by spinning at 14000 rpm for 1 minute. 

- When cultures exceed 1.5 ml, supernatants are removed and a fresh 1.5ml added to the pellet and respun to collect all the cells in the same tube. 

- Plasmid DNA is isolated using a Miniprep kit  according to the manufacturer’s instructions. 

- DNA isolation relies on alkaline lysis of bacterial cells, followed by adsorption of the plasmid DNA onto a 'silica membrane'. 

- Purified plasmid DNA is eluted with 50µl 'elution buffer' supplied by the manufacturer.


steps:
- It is required e.g. to screen plasmid DNA for the presence of a target insert or to prepare DNA bands for ligsation.

- DNA is digested using restriction enzymes. 

- For analysis, reactions are typically 10µl in volume and contain 1µl 10x bovine serum albumin (10mg/ml)
1µl 10x appropriate enzyme buffer (as recommended by the manufacturer)
2µl plasmid DNA sample (e.g, miniprep DNA) 
and 6µl MQ water. 

- The solution is mixed and 10U of each restriction enzyme is added, mixed again, spun briefly to recover the mixture which is then incubated at the appropriate temperature for the enzyme in use in a heating block for 1-2 hours. 

- For cloning these reactions are scaled up in volume and used 5-10U restriction enzyme per µg of DNA. 

- Digestion is checked by analysis of a small sample after 1 hour and if incomplete fresh enzyme is added and incubation is continued for a further hour. 

- The digest is analysed by agarose gel electrophoresis.

im1lab:
example of linearized plasmid vector [lane 1: 1kb DNA marker, lane 2: uncut, lane 3: cut 'linearized']


Technique:
SDS- PAGE polyacrylamide gel electrophoresis

steps:
- A 10% resolving gel is made containing 
4.6ml distilled water, 
10ml Ax2 ‘resolving buffer’  
5ml 40% acrylamide-bisacrylamide,
and 100µl 20% SDS. 

- Polymerisation of acrylamide is induced by addition of 
     200µl 10% ammonium persulphate 
and 50µl TEMED (N,N,N’,N’-Tetramethylethylenediamine) which acts as a catalyst for the polymerisation. 

- The resolving gel is immediately poured between the glass plates of minigel apparatus using a pipette to a level just below the expected level of the comb. 

- Water-saturated butanol is overlaid to exclude oxygen (an inhibitor of polymerisation) and to ensure a smooth surface to the gel. 

- The gel is left to set and the water-saturated butanol is discarded and the gel top was washed with distilled water. 

- A filter paper is used to dry the inside surfaces of the glass plates above the resolving gel but without touching the gel itself. 

-A 4% stacking gel is made containing 
3.75ml distilled water, 
5ml Bx2 ‘stacking buffer’, 
1ml 40% acrylamide-bisacrylamide, 
and 50µl 20% SDS. 

- Again, polymerisation is induced by addition of 
     100µl 10% ammonium persulphate 
and 30µl TEMED and then the gel was poured immediately on top of the resolving gel. 

- The comb is positioned in the stacking gel which then is left to set and the comb is carefully removed and the well pockets are washed in running buffer to remove acrylamide “tags”. 

- The gel tank is assembled and both reservoirs were filled with running buffer. 

- The protein samples are mixed with sample buffer (liquids samples were mixed with an equal volume of 2x sample buffer and solids 'pellet' are resuspended directly in 1x sample buffer) and boiled for 2 minutes for denaturation. 

- Prestained wide range (6-175 kDa) protein marker is boiled. 

- 15µl samples are loaded into the wells and one well is loaded with 10µl of the marker. 

- Electrophoresis proceeds at 150 volts for 1-2 hours. 

- The gel is carefully removed and stained with Coomassie blue stain  for about 1 hour on a desktop shaker and then is destained in several changes of destaining solution  until bands can be seen.


steps:
- The concentration of the expressed purified proteins is determined after purification by using the protein assay kit 'BioRad'. 

- Bovine serum albumin standard dilutions of 1.6, 0.8, 0.4, 0.2, and 0.1 mg/ml are prepared by 1:2 serial dilution of the first concentration (25µl each). 

- The sample is prepared by adding 2.5µl to 22.5µl distilled water. 

- Blank is a 25µl of distilled water. 

- 2ml reagent A and 40µl reagent S are mixed by vortex and 125µl is added to each of the blank, standard and sample tubes. 

- 1ml reagent B is added and mixed immediately to all tubes which are incubated at room temperature for 15 minutes. 

- The absorbance is measured at 750nm and results plotted to derive a standard curve using Microsoft  Excel program.

- the wave length used is determined in the used kit according to the produced colour.


steps:
- After running the protein samples on a polyacrylamide gel.
- equilibrate the gel in 3 changes of transfer buffer for 5 minutes each, by rocking it gently in a box at room temperature. 

- For each gel, 8 sheets of 3MM 'Whatman' paper and one sheet of Immobilon PVDF membrane (Millipore) are cut to the same size as the gel. 

- The 3MM papers are soaked in transfer buffer, while the membrane is socked first in methanol for 15 seconds and then in transfer buffer. 

- Arrange the blot layers in the electroblotting apparatus by stacking 4 pieces of 3MM paper on the bottom plate electrode of a  the semi dry blotter apparatus 'BioRad'. 

- These are followed by the membrane upon which the gel is carefully placed. 

- At all stages care must be taken to avoid trapping bubbles between layers of the stack.  

- Finally the remaining 4 pieces of 3MM paper are laid carefully on the top (one by one). 

- Any air bubbles that may have been present are removed by gentle rolling from the centre outwards with an empty 50 ml Falcon tube. 

- The upper electrode plate is put in position and the apparatus is closed. 

- Proteins are blotted from the top ‘negative electrode’ towards the bottom ‘positive electrode’ at 10-15V for 40 minutes with maximum current limit of 5.5Amp/cm2.


steps:
- The capacity of the membrane to bind protein non-specifically is blocked after blotting by overnight immersion of the blot in blocking solution containing 5% dried milk protein in 1x Tween 20 wash buffer at 4ºC. 

- The blot is then placed carefully inside a 50ml falcon tube ensuring that the protein side is facing the centre of the tube. 

-  5ml blocking buffer containing the appropriate dilution of  "1ry antibody" is then added, and the tube is rolled at the appropriate temperature for 1 hour on a Spiromix rotator. 

- Most antibodies are allowed to bind at room temperature, but in some cases suppliers specify 4ºC for this step in which case the rolling is carried out in the cold room. 

- The blot is then removed and washed twice in 1x tween-20 wash buffer for 30 seconds for each wash, and then twice for 10 minutes each time in the same buffer.

- It is then transferred to another 50 ml falcon tube containing 5mls of blocking buffer as above in which the appropriate dilution of the "secondary antibody" ( e.g. horse radish peroxidase (HRP) conjugate) is prepared. 

- Rolling incubation is continued at room temperature for 1 hour. 

- The blot is washed again as above before it is removed from the box, excess liquid is carefully removed .

- apply the chemiluminescent substrate on the ‘protein side’ of the membrane and distribute it by gentle pipetting. 

- The blot is removed, drain excess substrate and wrape the membrane in cling flim. 

- The wrap is placed in an X-ray cassette (protein side uppermost) and held in place with a tag of masking tape. 

- A fresh X-ray film (e.g. Fuji) is placed on the wrape and the cassette is closed. 

- An intial exposure is developed after 1-2 minutes to check the intensity of the signal and a fresh film is then inserted. 

- This is exposed for periods of a few minutes to an hour depending on the intensity of the bands generated in the intial exposure.


steps:
- sections of the DNA to be introduced are synthesised as oligomers in both positive and negative senses. 

- These are then annealed to create a double stranded molecule with protruding, single-stranded, sticky ends compatible with the restriction cleaved acceptor molecule into which the synthetic DNA is to be inserted. 

- A 100µl annealing reaction is used containing 
 10µl 10x annealing buffer, 
 45µl sense strand 
 and 45µl anti-sense strand 
in 1.5ml Eppendorf which is exposed to heat in boiling bath for 2 minutes and then transferred to 65ºC heating block for 5 minutes and finally transferred to 44ºC water bath for 2 hours. 

- The samples are left to cool overnight in the water bath after it is turned off. 

- The DNA strands are precipitated and resuspended in 90µl MQwater. 

- The strands are phosphorylated using 100µl reaction containing the 90µl DNA sample, 
10µl 10x kinase buffer, 
1µl 1 mM ATP 
and 2µl kinase enzyme (which catalyzes the transfer of a phosphate group from ATP to the free terminus of polynucleotides) and mixed. 

- The mixture is incubated at 37ºC for 45 minutes. 

- The annealed DNA strands are extracted from the solution by addition of equal volume (100µl) of phenol-chloroform-isoamyl alcohol to the sample followed by vortex mixing for 2 minutes.

-  Phases are separated by centrifugation (13000 rpm for 2 minutes) and the upper (aqueous) layer is harvested to a fresh microfuge tube. 

- The DNA is re-extracted, and precipitated and resuspended in 50µl MQ water.


steps:
- DNA strands are extracted from the solution by addition of 'equal volume' of phenol-chloroform-isoamyl alcohol to the sample followed by vortex mixing for 2 minutes.

-  Phases are separated by centrifugation (13000 rpm for 2 minutes) and the upper (aqueous) layer is harvested to a fresh microfuge tube.

- The main aim of the extraction is the 'exclusion of any enzymes' in the mixture which might harm the DNA.


im1lab:
Western blot showing expression of recombinant protein in yeast cells, lane 1: positive control showing beta galactosidase 119kDa band , and different pattern from other lanes, lanes 2,3,4,5,6,7: 6 different yeast clones showing the expressed protein with larger and smaller size bands indicating the glycosylated and degraded protein


steps:
- add 1ul alkaline phosphatase
- incubate 30 mins at 37 c
- then, add another 1ul alk phosphatase 
- incub 30 min at 37c


steps:
- Harvest the cells from 1 or more flasks by discarding the old medium, put new media and use sterile glass beads

- pellet the cells by centrifugation at 2500rpm for 5 min

- discard the supernatant

- resuspend the pellet in 1-2ml freezing medium per flask

- alliquot 1ml in each cryovial


steps:
- It is very important to do sequencing when you are doing a cloning experiment to characterize the final clone.
- When you clone a gene in an expression vector, you have to check the open reading frame 'ORF' with the promoter.
- you can use the chromas program to check the result of the sequencing file as it can open the chromatoogram files
- if you want to do alignment with another sequence (e.g. the expected sequence) you can use the CLC program 
- there is a link for both programs in the links

- link 1 shows a good sequence: i.e. only one clone with the correct expected sequence
- link 2 shows a bad sequence due to overlaping of 2 clones (i.e. showing 2 sequences over each other which cause the computer to write N opposite the confusing base)- To avoid this problem, you should be careful while selecting a single colony from a transformation plate to do plasmid isolation

-if the insert to be sequenced is ~500bp the sequencing can be done by using onl 1 primer, however in large inserts, both forward and reverse primers should be used.


Technique:
Alkaline lysis of bac cell wall for plas isolation

steps:
from:http://bitesizebio.com/articles/the-basics-how-alkaline-lysis-works/
(9-May-2011 / 4:45pm)

Alkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since. The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes.

1. Cell Growth and Harvesting

The procedure starts with the growth of the bacterial cell culture harboring your plasmid. When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium.

2. Re-suspension

The pellet is then re-suspended in a solution (normally called solution I, or similar in the kits) containing Tris, EDTA, glucose and Rnase A. Divalent cations (Mg2+, Ca2+) are essential for Dnase activity and the integrity of the bacterial cell wall. EDTA chelates divalent cations in the solution preventing Dnases from damaging the plasmid and also helps by destabilizing the cell wall. Glucose maintains the osmotic pressure so the cells don’t burst and Rnase A is included to degrade cellular RNA when the cells are lysed.

3. Lysis

The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). This process is called denaturation and is central part of the procedure, which is why it’s called alkaline lysis. SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process.

It is important during this step to make sure that the re-suspension and lysis buffers are well mixed, although not too vigorously (see below). Check out my article on 5 tips on vector preparation for gene cloning for more information and tips. Also remember that SDS and NaOH are pretty nasty so it’s advisable to wear gloves and eye protection when performing alkaline lysis.

4. Neutralization

Addition of potassium acetate (solution 3) returns decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal those huge gDNA stretches. This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.

5. Cleaning and concentration 

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, Rnase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. The most common methods are detailed in my article on 5 ways to clean up a DNA sample.


steps:
- While designing primers for sequencing, be careful (if you have multiple inserts in the cloning plasmid) at what step you need to do the sequence, after adding each insert as the primer sequence will differ, as adding other inserts will change the sequence of the whole plasmid map.
- sequencing PCR reaction is done using 1 primer only per reaction 
- Thermal cycler adjustment:
96 - 1min
25 cycles:
96 - 10sec
50 5sec
60 4 min
- After the PCR reaction, 
- use the columns to clean the PCR product
spin the columns 500 xg - 5 min
add the 10ul PCR product to the center of the gel in the column
spint the column again for 550 xg - 5 min
take the spin down, put in the sequencing plate


# methods precautions

precautions:
- Acrylamide is neurotoxic --> handle with gloves. It can be absorbed through unbroken skin. If skin comes in contact with acrylimide solution or powder, wash immediately with soap and a lot of water. Unpolymerized acrylimide should be polymerized with excess catalyst and disposed of with solid waste. DO NOT POUR UNPOLYMERIZED ACRYLIMIDE DOWN THE SINK.

- Amonium Persulfate should be made up fresh or used from a relatively fresh stock. It goes bad after a week or two in the refrigerator. It can be disposed of by dilution with water and pouring down the sink.

- TEMED should be stored in the refrigerator in dark glass bottles. A bottle should be good for about a year


# methods solutions

solution:
10x TBE Tris Borate EDTA Buffer: 1 litre


solution:
AX2 buffer (Resolving gel buffer): 500ml


# My links

description:
CLC Sequence viewer program, With sequences alignm


description:
EMBOSS explorer, Bioinformatics tools 'On line'


description:
NEB cutter, Know the restriction enzymes which cut


description:
Chromas program, Open chromatogram 'nucleotide seq


description:
Swiss pdb viewer 'spdbv', Do calculations on prote


description:
PDB 'Protein Data Base' Bank, Find a protein to op


description:
Identify a nucleotide sequence 'BLAST', You can en


description:
Clinical chemistry, Know different methods of solu


description:
Diagnosis of infectious diseases, organisms


description:
brain food for biologists, how substances work


# solution

prep:
0.6g  agarose
45ml  MQ water
5ml    10x TBE buffer
*dissolve in microwave at 3rd degree for 3 minutes
cool in water bath
add 2.5 ul ethidium bromide ‘10mg/ml stock’
pour in the gel cast ~ 40-45ml

prec:
NB: increasing the conc of the agarose (2%) if the target fragments are small [<500bp]


prep:
* Dissolving lyophilized primer:
- Make a freezer stock (which should be thawed relatively infrequently) at 100 uM concentration. 
- Add a volume of solvent e.g: TE (ul) equal to 10 times the number of nanomoles of DNA present in the tube (as noted on the spec sheet provided with the oligo) this will produce a stock solution 100uM. For example, dissolve 50 nmoles of oligo in 500 ul TE or pure water to make a stock 100 uM solution. 
- Make a working stock of 10uM
- Usually add 2ul of (10uM stock) to 50ul reaction --> final conc. = 0.4 uM 

Ours primer: Qty/tube = 7.8 nmoles -->
                                so, dissolve in 78 ul
*so, by logic resoning:
So, 7.8 nmole in 78 ul 
= 0.1 nmole in 1 ul
= 100,000 nmoles in liter
= 100 umole (um) in liter = 100uM


prep:
10mM     Tris-HCl (pH 9.0 at 25°C)
   50mM     KCl and 0.1%
   Triton® X-100

prec:
- check the strength 10x or 5x
- check if it contains MgCl2 or not 
[if not magnisium chloride should be added separately]


prep:
growth medium           10ml
FBS                                1ml
DMSO                            1ml


prep:
1x Tc100                              90ml
fetal bovine serum 'FBS'                                    10ml
glutamine                                1ml
penicillin/streptomycin             1ml

- warm to room temp. before use


prep:
Water                              4.6ml
Ax2 ‘resolving buffer’       10ml
Bx2 ‘stacking buffer’           -
Acryl:bis                             5ml
SDS  ‘20%’                       100ul
Ammonium persulphate   200ul
TEMED                              50ul


prep:
Water                              3.75ml
Ax2 ‘resolving buffer’           -
Bx2 ‘stacking buffer’          5ml
Acryl:bis                             1ml
SDS  ‘20%’                       50ul
Ammonium persulphate   100ul
TEMED                              30ul


prep:
- Prepare in falcon tube
- fill with buttanol till the middle
- add distilled water ‘MQ’ till you see 2 separate phases
- take from the upper phase which is the saturated buttanol, put over the resolving gel before drying to prevent aereation and formation of meniscus on the upper edge of the gel


solution:
AX2 buffer (Resolving gel buffer): 500ml

prep:
(0.75 M Tris. pH 8.8)

- Dissolve 45.4 g Tris in 500ml MQ water
- Adjust pH: 8.8 with concentrated HCl


prep:
363ml      MQ water
100ml     Methanol
37.5ml    Acetic acid 
1.25g      Coomassie brilliant blue stain (BioRad)


prep:
- Add the following to 800ml MQ water:
     3 g          Tris
    14.4 g      Glycine
- Adjust volume to 1 litre with Methanol

i.e. 25mM Tris, 192mM glycine, and 20% methanol


prep:
20ul      2x sample buffer      
10ul       sterile MQ water       
10ul       DNA marker 'ladder'


prep:
stock conc. = 10 mM
Final conc. 200 uM
To reach the final conc.
use the equation C1V1 = C2V2

eg: add 1ul (of the 10mM) to 50ul PCR reaction ? X 10,000uM = 200uM x 50
? = 10,000 / 10,000 = 1


prep:
- Dissolve the following in 800ml MQ water:
    24.2 g     Tris base 
    80 g        NaCl
- Adjust pH to 7.6 with HCl
- Add 10ml Tween-20, and mix well
- Make up to 1 litre with MQ water
- Store at 4◦C


prep:
5% w/v Marvel 'dried milk' in 1x TBS-Tween-20 (the wash buffer)


prep:
- Add the following to 800ml MQ water
      16g       Bactotryptone
      10g       Yeast extract
      10g        NaCl
- Adjust volume up to 1 litre with MQ water
- Sterilize by autoclaving


prep:
- Dissolve 1.2 g Bactoagar in 100ml 2x YT fluid medium by boiling.
- Cool to 50C


prep:
- 3.2 mM    Na2HPO4
          - 0.5 mM    KH2PO4
          - 1.3 mM    KCl
          - 135 mM   NaCl
          - 0.05%     Tween-20


prep:
40 mM    TrisHCl (pH 7.5)
     20 mM    MgCl2
     50 mM    NaCl


prep:
40 mM    Tris (pH 7.5)
20 mM    MgAc
0.1%       Bovine Serum Albumin


solution:
10x TBE Tris Borate EDTA Buffer: 1 litre

prep:
- Add the following to 800ml H2O:
    107.8g     Tris base 
     55g         Boric acid 
     9.3g        EDTA 
- Adjust volume up to 1 litre with sterile MQ water
*send to autocalve


prep:
25ml        10x TBE buffer     
 225ml      MQ water             
 12.5ul      Ethidium bromide (10mg/ml stock)

prec:
- ethidium bromide is carcinogenic

- dispose in charcol specified bottle


prep:
4ml      10x TBE buffer  
   4ml       Glycerol              
  12ml      MQ water          
  0.1 g      Bromophenol blue dye & xylene
               cyanol ff dye


prep:
- Add the following to 800ml MQ water
      3 g          Tris
     14.4 g      Glycine
      5ml         20% SDS
- Adjust volume up to 1 litre with MQ water
- pH should be 8.12, often does not need adjustment


prep:
10ml growth medium TC-100
1ml   FBS
1ml   DMSO

prec:
- freeze cells in cryovials
- freezing of cells must be slow
- put the cryovials inside polysterene box lined with wool-cotton
- place in -80c freezer, overnight
- next day, transfer the cryovials to the liquid nitrogen storage
- record the cells in the log book


prep:
0.25M Tris, pH= 6.8

dissolve 15.12 g   Tris 
in 500ml MQ water

adjust pH with conc HCl


prep:
40g     acrylamide
1.08g  bisacrylamide   [Ratio 37:1]
dissolve in 100ml water


prep:
0.1g    ammonium persulphate
1ml     water


prep:
363ml    MQ water
100ml    methanol
37.5ml    Glacial acetic acid
small amount of Commassie stain powder


prep:
363ml    MQ water
100ml    methanol
37.5ml    Glacial acetic acid

prec:
- destain for 1 hour and then change the destaining solution


solution:
LB Lauria Bertani medium with Carbenicillin

prep:
Antibiotic: Carbenicillin 
stock concentration: 
100 mg/ml or 50 mg/ml
Working conc: 100 ug/ml
so, to prepare 1000ml (1 liter) of LB medium use C1V1 = C2V2
50,000 ug/ml X ? = 100 ug/ml X 1000ml
? = 100 X 1000 / 50,000 = 2ml
so, Add 2 ml Carbenicillin stock (50mg/ml) to the 1 liter LB medium

prec:
Carbenicillin can be used instead of Ampicillin.
It is more stable than ampicillin


