|In vitro mutagenesis using Altered Sites |
1. Isolation of Single Stranded DNA
- Transform plasmid to be mutagenised into JM109. JM109 is endA1, recA1, gyrA96, thi, hsdR17(rk-, mk+), relA1, supE44, l-, d(lac-proAB), [F', traD36, proA+B+, lacIqdM15]. JM109 must be maintained on minimal plates supplemented with 1mM thiamine HCL to select for the F', required for formation of ssDNA (infection by M13).
Pick single colony and inoculate 2ml TYP. Grow overnight.
- 16g bacto-tryptone
- 16g bacto-yeast extract
- 5g NaCl
- 2.5g K2HPO4
- dH20 to 1 litre
- Take 100µl of overnight culture and inoculate 5ml TYP in a 50ml flask and incubate with vigorous shaking for 30 minutes.
- Infect culture with helper phage. For Promega supplied phage solutions, use 40ul, otherwise titre phage particles and infect at a multiplicity of infection of 10. Add 50µl of 2M K2HPO4 and continue incubation for 6 hours to overnight.
- Collect the culture supernatant by centrifugation at 12000g for 15 minutes. Pour supernatant into a fresh tube and repeat centrifugation.
- Precipitate the phage by adding 0.25 volumes of phage precipitation solution. Leave on ice for 30 minutes and spin at 12000g for 15 minutes. Discard supernatant and resuspend pellet in 400ul TE and transfer to an ependorf tube.
Phage precipitation solution:
- 3.75M ammonium acetate pH 7.5
- 20% PEG-8000
- Extract once with chloroform:isoamyl alcohol (24:1).
- Extract with TE saturated phenol/chloroform. Repeat extraction until there is no visible material at the interface.
- Precipitate ssDNA with 0.5 volumes of 7.5M NH4Ac and 2 volumes EtOH. Wash pellet with 70% EtOH.
- Resuspend the ssDNA in 20ul dH20 or TE.
- Analyse 2ul sample by electrophoresis (M13KO7 ssDNA runs at 8.7Kb and R408 at 6.4Kb). The presence of helper phage DNA does not affect mutagenesis.
2. Phosphorylation of Oligo
- Resuspend 1ug of oligo in 17.5 ul dH2O. Add 2.0ul of 10x kinase buffer and 5U T4 polynucleotide kinase. Incubate at 37°C for 30 minutes.
10x kinase buffer:
- 500mM Tris-Cl pH7.5
- 100mM MgCl2
- 50mM DTT
- 1mM spermidine
- 10mM ATP
- Incubate the reaction at 70°C for 10 minutes.
- Add 80µl dH2O (to give final concentration of 10ng/µl), or 430µl dH2O (to give final concentration of 2.2 ng/µl).
3. Annealing reaction and mutant strand synthesis
- Add to an ependorf: 100ng ssDNA
- 1ul 2.2ng/µl phosphorylated ampicillin repair oligo
- 1ul 10ng/µl phosphorylated mutagenic oligo
- 2ul 10x annealing buffer
- dH20 to 20µl
10x annealing buffer
- 200mM Tris-Cl pH 7.5
- 100mM MgCl2
- 500mM NaCl
- Heat to 70°C, and allow to cool to room temperature over 15-20 minutes.
- Place on ice and add 3ul 10x synthesis buffer, 10U T4 DNA polymerase, 2U T4 DNA ligase and 5ul dH2O. Incubate at 37°C for 90 minutes.
10x synthesis buffer:
- 100mM Tris-Cl pH 7.5
- 5mM dNTPs
- 10mM ATP
- 20mM DTT
4. Transformation into ES1301 (lacZ53, mutS201::Tn5, thyA36, metB1, deoC, in (rrnD-rrnE)
ES1301 is a mismatch repair minus strain. Use of this mutS strain prevents repair of the newly synthesised unmethylated strand, leading to high mutation efficiencies. ES1301 is also recA+ and therefore inserts with repetitive sequences may be unstable. ES1301 is KanR due to the Tn5 transposon.
- To the synthesis reaction, add 1µl 10mg/ml tRNA, 0.1 vol 3M NaAc and 2.0 vol EtOH. Spin 15 minutes. Wash pellet in 70% EtOH, then resuspend in 10µl dH2O.
- Transform into electrocompetent ES1301.
- Add cells to 1.0ml SOC broth and incubate for 30 minutes at 37°C.
- Add to 3ml LMM medium containing 170µg/ml ampicillin and 16.7µg/ml tetracycline in a 50ml falcon tube and incubate with shaking for 12 to 14 hours.
5. Transformation into DH5a
- Miniprep plasmid from 1.5 ml of above culture.
- Transform 0.05 to 0.10 µg of plasmid into DH5a cells and plate on medium containing 125 µg/ml ampicillin and 12.5 µg/ml tetracycline.
- Pick single colonies and grow up for analysis in medium containing 100µg/ml amp and 12.5 µg/ml tet.
|- ||Best yields of ssDNA are observed when infecting JM109 cells with R408 helper phage. |
|- ||Oligo concentration is dependent on length. Figures given above are for a 23mer mutagenic oligo. The molar ratio of ampicillin repair oligo : mutagenesis oligo : template should be 5 : 25 : 1 |
|- ||pALTER-Control has a defective LacZ gene which should be repaired using the control oligonucleotide as a control for the efficiency of mutagenesis. |