In vitro mutagenesis using Altered Sites

Ⅰ.Isolation of Single Stranded DNA

1.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.

TYP medium

16g bacto-tryptone

16g bacto-yeast extract

5g NaCl

2.5g K₂HPO₄

dH₂O to 1 litre

2.Take 100µl of overnight culture and inoculate 5ml TYP in a 50ml flask and incubate with vigorous shaking for 30 minutes.

3.Infect culture with helper phage. For Promega supplied phage solutions, use 40μl, 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.

4.Collect the culture supernatant by centrifugation at 12000g for 15 minutes. Pour supernatant into a fresh tube and repeat centrifugation.

5.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 400μl TE and transfer to an ependorf tube.

Phage precipitation solution:

3.75M ammonium acetate pH 7.5

20% PEG-8000

6.Extract once with chloroform:isoamyl alcohol (24:1).

7.Extract with TE saturated phenol/chloroform. Repeat extraction until there is no visible material at the interface.

8.Precipitate ssDNA with 0.5 volumes of 7.5M NH4Ac and 2 volumes EtOH. Wash pellet with 70% EtOH.

9.Resuspend the ssDNA in 20μl dH₂O or TE.

10.Analyse 2μl sample by electrophoresis (M13KO7 ssDNA runs at 8.7Kb and R408 at 6.4Kb). The presence of helper phage DNA does not affect mutagenesis.

Ⅱ.Phosphorylation of Oligo

1.Resuspend 1ug of oligo in 17.5 μl dH₂O. Add 2.0μl of 10x kinase buffer and 5U T4 polynucleotide kinase. Incubate at 37℃ for 30 minutes.

10x kinase buffer:

500mM Tris-Cl pH7.5

100mM MgCl₂

50mM DTT

1mM spermidine

10mM ATP

2.Incubate the reaction at 70℃ for 10 minutes.

3.Add 80µl dH₂O (to give final concentration of 10ng/µl), or 430µl dH₂O (to give final concentration of 2.2 ng/µl).

Ⅲ.Annealing reaction and mutant strand synthesis

1.Add to an ependorf: 100ng ssDNA

1μl 2.2ng/µl phosphorylated ampicillin repair oligo

1μl 10ng/µl phosphorylated mutagenic oligo

2μl 10x annealing buffer

dH₂O to 20µl

10x annealing buffer

200mM Tris-Cl pH 7.5

100mM MgCl₂

500mM NaCl

2.Heat to 70℃, and allow to cool to room temperature over 15-20 minutes.

3.Place on ice and add 3μl 10x synthesis buffer, 10U T4 DNA polymerase, 2U T4 DNA ligase and 5μl dH₂O. Incubate at 37℃ for 90 minutes.

10x synthesis buffer:

100mM Tris-Cl pH 7.5

5mM dNTPs

10mM ATP

20mM DTT

Ⅳ.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.

1.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.

2.Transform into electrocompetent ES1301.

3.Add cells to 1.0ml SOC broth and incubate for 30 minutes at 37℃.

4.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.

Ⅴ、Transformation into DH5a

1.Miniprep plasmid from 1.5 ml of above culture.

2.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.

3.Pick single colonies and grow up for analysis in medium containing 100µg/ml amp and 12.5 µg/ml tet.

NOTES:

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au

David Bowtell PMCI October 1998