Standard Techniques for Molecular Biology
Isolation of Genomic DNA (bacteria)
Quick and Dirty
Scratch a pure colony of your bacteria freshly grown on a (selective) agar plate and resuspend it in 100 µl 10 mM Tris pH 8
Boil for 10 min and cooled down.
Break up cells by pipetting 20 times through a yellow pipette tip - hoping this will nor shear the genomic DNA too much.
Using any commercially available kit for genomic DNA isolation.
Usually they work very well while the "quick & dirty-method" frequently results in a big waste of time and reagents.
Primers
General considerations for primer design:
- both primers should have a similar annealing temperature
- high annealing temperatures increases reaction specifity
- the primer should have at 3' 1-2 purine bases for a stable annealing for the DNA-polymerase
- the primer should not have at 3' more than 2 purine bases to avoid to stabilise mispriming
- the primer should not have more than 4 same bases in series to avoid mispriming
- the primer should not form secondary structures, e.g. hairpins
- the primer(s) should not have sequences complementaries to avoid primer dimers
- when introducing restriction sites, add 3 nt additionally as restriction enzymes do nut cut at very ends. This may cause problems with mispriming. Moreover, Taq frequently keeps attached to the DNA inhibiting the cutting by restriction enzymes
- primers do not have a 5'-phosphate. For blunt end ligation into a dephosphorylated vector, they have to be introduced (polynucleotide kinase, PNK)
For cloning properties with primers of max. 30 nt a 0.01 µmole scale is sufficient
The primers can be dissolved in 1x TE at a stock conc. of 100 pmol/µl (= 100 µM) and stored at -20 °C.
For the working soln the stock soln should be diluted 1:10 (i.e. 10 pmol/µl) with 10 mM Tris (pH 8). Do not dilute with TE as EDTA will chelate Mg and thus inhibiting the DNA-polymerase
PCR
THE POLYMERASE CHAIN REACTION IS USED TO AMPLIFICATE TEMPLATE DNA in-vitro and is performed basically in 3 steps, which are repeated about 30 times:
1) Denaturation of the double stranded DNA to single strand DNA
- 20 - 30 s @ 94 ºC. Depends on the GC content of the template
- additives like glycerol may facilitate the denaturalisation
2) Annealing of the primers to the ssDNA
- 30 - 40 s @ 45 - 65 ºC. Temp. depends on the primer length and should be 5 ºC lower than Tm.
- melting temp. (Tm) can be calculates as Tm [°C] = (G + C) * 4 + (A + T) * 2
- too low annealing temp. may generate non specific products
3) Elongation
- 30 - 180 s @ 72 ºC. Extension time depends on length of the amplifying PCR fragment and can be calculated with 1 kbp/min (for Taq) and 0.5 kbp/min for a proof-reading DNA-polymerase
- some DNA-polymerases prefer higher temperatures; for amplifying long fragments usually lower temperatures (68-70 ºC) are used
A nice animation was made by Lodish et al. ©:
Additionally:
A) Initial denaturation: 2 - 3 min @ 95 ºC. For GC-rich genomes up to 10 min denaturation. Prolonged denaturation (> 3 min) or high temp. (> 95°C) inactivates Taq rapidly (=> hot start)
B) Final polymerisation (5 min @ 72 ºC) to fill-in the protruding ends of newly synthesised PCR products and to add an additional dA to the 3'-end of the PCR product (for TA-cloning)
C) Cooling down (hold 5 - 10 ºC) to inhibit unspecific DNA-polymerase activity
Factors influencing PCR:
Template
- purity of template: template solution may contain inhibitors of DNA polymerase (salts, detergents solvents - see Tab.). EtOH precipitation usually is sufficient for cleaning DNA
| substance |
SDS |
phenol |
ethanol |
isopropanol |
EDTA |
NaAc |
NaCl |
| inhib. conc. |
> 0.005 % |
> 0.02 % |
> 1 % |
> 1 % |
> 0.5 mM |
> 5 mM |
> 25 mM |
- when using long templates, special care have to be taken to avoid shearing of the DNA
- correct concentration of template DNA (frequently too much vector and not enough genomic DNA is used): 10-100 pg plasmid DNA, 10-100 ng bacterial genomic DNA, 0.1-1 µg human genomic DNA
Primers
- for fragments up to 5 kb primer length of 18 - 25 nt are recommended
- primer concentration should be 0.1 - 1.0 µM. Higher concentrations may yield in non specific product
- for general considerations of Primer design, see above
Mg2+ (standard: 1.5 - 2.0 mM)
- Mg2+ affects the annealing process and stabilises the DNA-polymerase complex
- optimal concentration depends on dNTP and template concentration and if EDTA containing soln are added
- high Mg2+: low fidelity, fast, non-specific primer binding
- low Mg2+: higher fidelity, slower, low yield
Ions
- K+ stabilizes primer annealing (= less specific).
- NH4+ destabilises primer annealing, specially when mismatched (= more specific).
dNTP
- avoid frequent freeze-thaw cycles
- recommend concentration is 200 µM (each). For long PCR fragments more (influencing Mg2+)
- lower concentrations (20-50 µM) rises up the fidelity of Taq and higher proceeding
Enhancer
- DMSO (2 - 10 %) or glycerol (1 - 5 %) can be used for better denaturalisation, especially for GC-rich templates. This decreases also the primer annealing temperature. As DMSO inhibits the Taq, Taq concentration. should be augmented
- BSA (up to 0.8 µg/µl) binds PCR inhibitors and stabilises the DNA-polymerase
DNA-polymerase
Usually a Taq-polymerase is used for PCR. This enzyme is thermostable (t1/2 @ 95 °C > 40 min) and has a high processivity (1-2 kb/min). For cloning purpose it should be considered, that it produces 3'-dA overhangs (TA-cloning) and has no 3'-5'-exonuclease activity (proof-reading) resulting in an error rate of 2.2*10-5 (= 1 error each 4.5*104 nt).
Therefore for cloning experiments frequently DNA-polymerases with proof-reading are used like the Pfu. This enzyme is highly thermostable (95% active after 2 h @ 95°C) and due to the 3'-5'-exonuclease activity it has a 10x higher fidelity than Taq: error rate is 2.6*10-6 (= 1 error each 3.8*105 nt). The consequences of this are, that Pfu produces blunt end PCR-products and is slower than Taq (500 bp/min). Be careful: the 3'-5'-exonuclease activity may degrade the primers at RT (when preparing the PCR-Mix).
Cycles
More than 30 cycles are not recommended as the Taq decays and may generate artefacts
Hot start PCR
Sometime necessary when using genomic DNA as template: due to wrong annealing of the primers to the template at low temperature and still some activity of the Taq-polymerase, DNA synthesis initiate from theses misprimed DNA duplexes creating incorrect substrates for the future rounds of amplification. This can be avoided by adding Taq-polymerase after the initial denaturation step. More sophisticated solutions are incorporation of Taq into wax beads or antibodies.
Mastermix
To avoid much pipetting for preparing a PCR it is useful to prepare a "MasterMix", which contains all but the template, primers and DNA-polymerase.
| Mastermix |
Pfu |
Taq |
| |
Volume |
Final concentration |
Volume |
Final concentration |
Volume |
Final concentration |
10x buffer
(with MgSO4) |
100 µl |
20 mM Tris-HCl (pH 8.8)
10 mM (NH4)2SO4
10 mM KCl
0.1 % Triton X-100
0.1 mg/ml BSA
2 mM MgSO4 |
100 µl |
75 mM Tris-HCl (pH 8.8)
20 mM (NH4)2SO4
0.01 % Tween 20 |
100 µl |
100 mM Tris-HCl (pH 8.8)
500 mM KCl
0.8 % Nonidet P40 |
| MgCl2, 25 mM |
... |
... |
80 µl |
2 mM MgCl2 |
60 µl |
1.5 mM MgCl2 |
| dNTP, 2 mM |
100 µl |
0.2 mM (each) |
100 µl |
0.2 mM (each) |
100 µl |
0.2 mM (each) |
| A. dest. |
600 µl |
|
520 µl |
|
540 µl |
|
This gives a volume of 800 µl, thus missing 200 µl (or 25 %) to have 1 ml.
Means to 24 µl Mastermix have to be added 6 µl:
| PCR-Mix |
Volume |
Final concentration |
| Mastermix |
24 µl |
1x |
| Primers, 10 pmol/µl |
1.5 µl (each) |
= 500 nM (each) |
template:
- genomic DNA (~ 100 ng/µl)
- plasmid DNA (~ 400 ng/µl, Midi-prep) |
~ 1 µl
~ 1 µl (dil. 1:100 !) |
~ 100 ng
~ 4 ng |
DNA-polymerase:
- Pfu (2.5 U/µl)
- Taq (5 U/µl) |
0.50 µl
0.25 µl |
1.25 U
1.25 U |
| adjust with A. dest. to |
6 µl |
= 30 µl |
Gel electrophoresis
SEPARATING NEGATIVELY CHARGED DNA IN A ELECTRIC FIELD BY THE FRAGMENT SIZE.
Usually agarose concentrations between 0.5 - 2.0 % are used, depending on the fragment size to be separated. The 2 most common buffers are:
a) 0,5x TBE:
- high buffer capacity (can be used at higher voltage = faster)
- small fragments (< 2 kb) are better separated
- DNA purification out of a gel is less efficient
b) 1x TAE
- low buffer capacity (can be used only at low voltage = slow)
- big fragements (> 2 kbp) are better separated
- DNA purification out of a gel has good efficiency
We use a cheap alternative, 0.5x SB-buffer (sodium borate) to achieve a better separation of small fragments at high voltage.
Our standard size markers are: λPstI (5-10 µl), which can be easily self made, 1 kb-ladder (5 µl) or 50 bp-ladder (5 µl).
The loading dye (storage at - 20 °C) contains:
- 60 % glycerol that the sample enters into the wells and
- 2 blue stains to estimate the separation of the DNA: The co-migration of xylene cyanol FF is at ~ 4000 bp and of bromophenol blue is at ~ 300 bp.
| Agarose conc. |
SB |
TBE |
TAE |
| 0.7 % |
0.3 - 4 kb |
0.5 - 8 kb |
0.8 - 15 kb |
| 1.0 % |
0.2 - 3 kb |
.. |
0.5 - 10 kb |
| 1.5 % |
0.05 - 1.5 kb |
.. |
0.4 - 6 kb |
| 2.0 % |
.. |
0.05 - 2 kb |
0.3 - 3 kb |
For the Owl Easycast B2 (14 x 12 cm); distance between electrodes ~ 20 cm:
Gel height: 50 ml = 0.30 cm; 100 ml = 0.6 cm; 150 ml = 0.9 cm
Run: 5 min @ 50 V (2.5 V/cm), 5 min @ 100 V (5 V/cm) and afterwards @ 150 V (7.5 V/cm), with high Ampere & high Watt settings to achieve constant voltage. Using SB-buffer the gel will not heat up.
For 6 cm (half gel): ~ 60 min, for 12 cm (full gel): 90 min.
Staining
The DNA in the agarose gels is usually stained with Ethidium bromide, which is a sensitive (5 ng) and cheap procedure. Another dye is SYBR Green/Gold (Molecular Probes), which is more sensitive (0.02 ng), may be less toxic but much more expensive. Not very comon but possible is Methylene blue (200 µg/ml): this not not toxic, not very sensitive (40 ng), needs a destaining step but does not need UV-illumination.
Ethidium bromide (EtBr) intercalates between bases of the DNA and is therefore very toxic. Nitrile gloves have to be used for personnel protection, moreover, adsorbent paper should be placed around the working area.
Add 10 µl EtBr (10 mg/ml) into 200 ml A. dest. (= 0.5 µg/ml) and stain the develloped gel for 20-30 min. Pour the staining solution back into a dark plastic container for later reuse and destain the gel for 2 x 5 min with A. dest.
For detoxification of EtBr containing solutions pour them over a filter containing activated carbon. Gels, paper, gloves, filters with activated carbon has to be collected seperately.
Photo documentation
As the DNA still diffuses within the gel, the staining and photodocumentation process must be carried out directly after the electrophoresis.
Place the stained gel onto a UV-transilluminatior and turn it on.
Use λ = 254 nm for introducing DNA damage
λ = 302/312 nm for documentation purpose (excitation of EtBr with less photo-nicking & dimerisation) and
λ = 365 nm for ...?
Long time exposures should be avoided as the fluorescence signal disappears and damages into the DNA will be introduced (specially problematic for cloning purpose).
The excitation for ErBr stained DNA is at 302 nm, the emission maximum is at 615 nm. Therefore the camara needs a filter to eliminate the excitation light and which passes only the emission light.
Settings for a standard digital CCD-camara:
B/W (grey-scale), f3.5-4.5, 4/2/1 s, ISO 100/200/400, wide angle
(high ISO with short exposure for cloning purpose, low ISO with long exposure for documentation purpose only)
Exposure MANUAL, Metering CENTRE WEIGHTED
Focus AUTO, Macro OFF, AF-Light OFF, Flash OFF, White balance: AUTO/custom, Image size: 2-5 MPix
depending on the equipment: Image stabilisation OFF, Teleconverter ON
ISOLATE DNA OUT OF AGAROSE GELS OR DESALT AND PURIFTY DNA FOR DOWNSTREAM REACTIONS.
This is a general description of the principal; different DNA-purification kits use the same procedure, only the names and quantities may vary.
Cut the band of interest from a agarose gel - as sharp as possible (~ 300 mg)
add ~ 3 vol. of Solubilisation-buffer (contains chaotropic salts to dissolve the agarose)
gels made with TAE usually dissolve easier than those from TBE
heating up to 50 °C accelerates this step. The agarose has to be dissolved completely
for fragments > 4 kbp add additionally 2 vol. A. dest.
in some kits the solutions contain a pH indicator (pH < 7): A colour/pH change can be adjusted by adding 5 - 10 µl 3 M NaOAc
- Add Adsorption-buffer (contains e.g. diatomic earths to adsorb the DNA): 10 µl for each 2 µg DNA or
- Pour the solution on a spin-column (contains e.g. diatomic earths to adsorb the DNA)
Centrifuge 1 min at 12,000 g, remove supernatant / flowthrough
Wash pellet / spin-column with 500 µl Wash-buffer to remove residual agarose (for desalting DNA this is not necessary)
Wash pellet / spin-column 2× with 500 µl Wash-buffer (contains EtOH to removes salts, proteins etc, does not resolve DNA)
- Air dry pellet to remove residual EtOH. Do not over dry pellet
- Centrifuge spin-column for 1 min at max. speed for to remove residual EtOH
Elute DNA with 20 µl 10 mM Tris, pH 8, incubating for 10 min at RT (fragments > 4 kbp at 50 ºC).
Centrifuge and transfer supernatant / flow through into new Eppendorf-tube
Repeat elution with another 20 µl and combine the supernatants
Electrocompetent cells
OBTAIN CELLS WHICH CAN RECEIVE DNA BY ELECTROPORATION. The cells have to be in salt free liquid to prevent a short circuit during the electroporation. Like this the cells are very sensitive and all steps have to be performed on ice.
Inoculate a overnight culture of Escherichia coli DH5α (or other strain) in 3 ml LB media
Inoculate 2 x 100 ml LB with each 1.5 ml overnight culture in a 250 ml baffled Erlenmeyer
Incubate cells until OD600 = 0.6 (about 4 h at 37 ºC shaking with 200 rpm)
Harvesting cells (centrifuge 10 min, average 7,000 g, 4 ºC)
Wash cells twice with 25 ml ice cold sterile A. dest. Centrifuge as above.
Wash cells twice with 20 ml 10 % ice cold sterile glycerol. Centrifuge as above.
Resuspend cells in 1 ml ice cold GYT (softly pipetting)
Transfer 120 µl cells into sterile Eppendorf tubes
Shock freeze in liquid nitrogen (recommended)
Storage of electrocompetent cells at -80 ºC (recommended)
Ligation
LIGATION OF A DNA FRAGMENT INTO A VECTOR.
- blunt end or TA-ligation can be enhanced by long incubation at low temp. (16 °C overnight)
- for blunt ligation, the vector should be dephosphorylated to avoid self ligation (bacterial alkaline phosphatase BAP is better than CIAP). Heat inactivate the phosphates. Remember, the primers do not have 5'-phosphates. See also primer design
- the vector-insert ratio should be 1 : 3-10 (sticky ends ... blunt ends)
- as salts, EDTA inhibit T4 ligase, in those cases EtOH precipitation is recommended
- PEG 4000 increases ligation rate for blunt ended DNA
- PEG may reduce the electroporation efficiency (especially by prolonged ligation reaction or when heat inactivated) and should be removed (EtOH precipitation)
Easily 3 ligations can be realised in parallel:
| |
I |
II |
III |
| 10x ligase buffer (contains ATP) |
2.2 µl |
| PEG 4000 |
1.0 µl (only for blunt end ligation or TA cloning) |
| vector |
0.5 - 2.0 µl |
| insert |
1.0 - 15.0 µl |
| A. dest. |
adjust final vol. to 22 µl |
| T4 DNA ligase |
1 µl |
Ligation overnight at 16 ºC
Inactivation of T4-DNA ligase by heating for 20 min at 65 °C
Add 80 µl A. dest., 300 µl 96 % ethanol and 1 µl glycogen (helps precipitating the DNA), mix well
Precipitation of DNA for 2 h at -20 ºC (to remove salts of the buffer and the ligase, which interfere with the subsequent electroporation)
Centrifuge 20 min at 16,000 g
Remove supernatant and air-dry pellet (not always visible). Do not over dry pellet
Redissolve DNA in 5 µl A. dest. and store on ice for the subsequent electroporation
Blunt end Ligation into pCR-Script
1) Digestion with Eco32I (bunt end) and dephosphorylation (FastAP) of the vector pCR-Script:
| Volume |
Item |
Final |
| 103 µl |
pCR-Script (0,5 µg/µl) |
50 µg |
| 12 µl |
10x R+-buffer |
1x |
| 5 µl |
Eco32I (10 U/µl) |
50 U |
| |
- incubate 2 h @ 37 °C |
|
| 2 µl |
FastAP (1 U/µl) |
2 U |
| |
- incubate 30 min @ 37 °C |
|
| |
- inactivate enzymes by heating for 10 min @ 75 °C |
|
2) Phosphorylation of the PCR-product
As the vector for blunt end ligation was dephosphorylated, and PCR-products do not have 5'-phosphates, now this has to be phosphorylated using a Polynucleotidkinase, PNK.
| Volume |
Item |
Final |
| 17 µl |
PCR product |
|
| 2 µl |
10x T4 Ligase buffer
- do not use PNK-buffer! - |
0.5 mM ATP |
| 1 µl |
PNK (10 U/µl) |
1 U |
| |
- incubate 30 min @ 37 °C |
|
| |
- inactivate PNK by heating for 15 min @ 70 °C |
|
TA-cloning
When using Taq-polymerase in the PCR, the PCR-product will receive a final dA-overhang thus it can not be ligated into blunt end vectors. On the other side, when the vector carries a dT-overhang the ligation can be facilitated compared to blunt end ligations.
| Volume |
Item |
Final |
| 25 µl |
pCRS Eco32I cutted |
~ 12,5 µg |
| 1.8 µl |
25 mM MgCl2 |
1.5 mM |
| 3 µl |
10x Taq-buffer |
1 x |
| 0.3 µl |
100 mM dTTP |
1 mM |
| 0.5 µl |
Taq-polymerase (5 U/µl) |
2.5 U |
| |
- incubate 1 h @ 65 °C |
|
| |
Alcoholic precipitation:
- Add 80 µl A. dest., 300 µl 96 % ethanol and 1 µl glycogen, mix well
- Precipitate DNA for 2-4 h at -20 ºC
- Centrifuge 20 min at 16,000 g
- Remove supernatant and air-dry pellet (not always visible). Do not over dry pellet
- Redissolve DNA in 30 µl TE |
|
Electroporation
TRANSFER VECTOR INTO COMPETENT BACTERIA, generating a GMO (genetically modified organism).
All steps are carried out on ice and as fast as possible
Thaw up electrocompetent cells (stored at -80 ºC) on ice
Transfer 40 µl electrocompetent cells to 5 µl ligation mix. Mix softly by pipetting (cutted tip)
Transfer both into a cold electroporation cuvette (usually 10 mm gap)
Pulse cells with 12,500 V/cm (for a 10 mm gap: 1,250 V)
Add immediately 1 ml LB
Transfer all into an Eppendorf tube and incubate cells for 60 min that the cell initiate expressing the resistance gene of the vector
Plate 50 µl cells on selective agar plate (according to the resistance gene)
Centrifuge cells 1 min at 10,000 g, remove nearly all supernatant (e.g. by pipetting)
Resuspend cells and plate all on another selective agar plate
Incubate plate overnight at 37 ºC
The manual of the electroporator (Eppendorf) recommends a 17,000 V/cm pulse for E. coli.
The electroporation cuvettes are to be sterilised with 70 % EtOH (overnight to kill the GMO), washed with 1 M acetic acid (to destroy DNA with acid), washed thoroughly with A. dest. (to rise up the pH again) and finally sterilised with 70 % EtOH before the next use.
Miniprep
ISOLATION OF PLASMID DNA OUT OF BACTERIA.
- High copy vectors: 5 µg DNA/ml LB
- Low copy vectors: 0.5 µg DNA/ml LB
Transfer from 2 ml overnight culture 1.5 ml into a Eppendorf-tube
Centrifuge 1 min at 10,000 g and remove supernatant
Resuspend cells in 100 µl Sol A and add 0.1 µl RNase A
Add 150 µl Sol B and mix softly to not share chromosomal DNA
Incubate 30 min at RT
Add 200 µl Sol C and mix softly to not share chromosomal DNA
Incubate 20 min at 4 ºC for precipitating proteins and chromosomal DNA
Centrifuge 10 min at 16,000 g and transfer 400 µl supernatant into new Eppendorf tube
Add 450 µl isopropanol and incubate 15 min at 4 ºC to precipitate plasmid DNA
Centrifuge 10 min at 16,000 g and remove supernatant
Wash the pellet with 600 µl 70 % ethanol and remove supernatant
Air-dry pellet: do not over dry the pellet (difficult to resolve DNA)
Resolve pellet in 20 - 40 µl TE
Recommendation:
After transfering the cells for a Miniprep, do not throw away the cells which rest in the test tube
If a Miniprep is positive, add into the respective test tube 2 ml (selective) media and let them grow overnight
Perform a cryoconservation of these cells thus you can use them directly for reproducing the respective vector, when it is necessary
ISOLATION OF HIGHLY PURIFIED PLASMID DNA OUT OF BACTERIA.
Plasmid DNA can be highly purified by phenol-isoamylalcohol extraction and/or CsCl centrifugation. Nowadays more common are kits for isolationg plasmid DNA w/o these steps.
Each supplier has its own (secret) recipie but generally contain the following steps:
- Centrifuge cells of an overnight culture, discard medium and resuspend in buffer with RNase A
- Add alcaline lysis buffer (containing detergents like SDS), incubate for to lyse the cells
- Do not vortex etc. for not shearing genomic DNA
- Add precipitation buffer (containing NaOAc) for neutralising lysis buffer and precipitating proteins, genomic DNA, cell wall components etc.
- Centrifuge and transfer supernatant onto a column (diatomaceous earth or anion exchange) which adsorbs specifically DNA
or transfer supernatant into a new tube and add adsorption matrix
Wash cell content with EtOH containing washing buffer away
Elute DNA with TE.
Yield: 100-350 µg DNA per 25 ml cell culture (high copy plasmid)
DNA concentration
DERMINE THE PURITY AND CONCENTRATION OF dsDNA
This is only useful for highly purified genomic or plasmid DNA. DNA from home-brew quick and dirty extraction methods contain too much contamination (protein, RNA).
Dilute DNA 1:100 with TE-buffer (10 µl DNA + 990 µl TE)
Determine the absorbance in a quartz cuvette at λ 260 (DNA), 280 (protein) and 320 (background) nm
Concentration: c = (A260 - A320) * 50 [extintion coefficient dsDNA] * 100 [dilution factor] = µg/ml
Purity: (A260 - A320) / (A280 - A320) = 1.6 - 1.9 (for pure dsDNA)
Troubleshooting:
A260/A280-ratio too low
-> sample was diluted with water instead of TE
A260/A280-ratio too high
-> DNA is contaminated with RNA (improve RNase A treatment)
Cryoconservation
LONG TERM STORAGE OF BACTERIA AND YEAST
Inoculate 2 ml (selective) media and incubate overnight
Transfer cells into 1.5 ml Eppendorf tube
Centrifuge 1 min at 7,000 g and remove supernatant
Resuspend cells in 1000 µl (selective) media (pipetting)
Add 500 µl Cryoconservation solution (containing ~ 65% Gro)
Vortex well and congelate immediately at -20 °C or better at -80 °C
Recommendation:
After transfering the cells for a Miniprep, do not throw away the cells which rest in the test tube
If a Miniprep is positive, add into the respective test tube 2 ml (selective) media and let them grow overnight
Perform a cryoconservation of these cells thus you can use them directly for reproducing the respective vector, when it is necessary
To reactivate cell, do not thawn them up:
Add 2 ml (selective) media into a test tube
Enter with a tooth pick into the frozen glycerol conservation and
put it into the prepared test tube
Incubate it overnight
Escherichia coli DH5α
MOST COMMON STRAIN FOR BACTERIAL TRANSFORMATION. DERIVED FROM NON-PATHOGENIC K12-STRAIN
Genotype: F- thi-1 endA1 hsdR17 recA1 deoR lacZδM15 glnV44 relA1 gyrA96
- F-: Does not carry F+-plasmid for conjugation (biosafety)
- thi-1: thiamine auxotroph (biosafety)
- endA1: Does not produce endonuclease I (no digestion of your favorite plasmid)
- hsdR17: Does not produce EcoKI (no digestion of your favorite plasmid)
- recA1: Inhibits homologous recombination
- deoR = nupG: permits uptake of large plasmids
- lacZΔM15: deletion of aa 11-41 of the β-galactosidase gene (α-complementation for blue-white screening)
- glnV44: Suppression of the UAG-stop codon and replacement with Gln (necessary for some bacteriophages)
- relA1: Permits RNA production without protein synthesis
- gyrA96: Nalidixic acid resistance
Last modified: 01.11.2013