Difference between revisions of "PhiX:Mutagenesis"
(Created page with 'COMPARE to my protocol Primer phosphorylation with T4 PNK: we phosphorylize 200 pmol of primer in 2ul reaction mixture Annealing: 10 pmol primer, 0.5 p mole ssDNA in 10ul PE1 b…') |
|||
| Line 11: | Line 11: | ||
We then add ATP to 1 mM, nucelotides to 0.5 mM, four units of T4 ligase, and 2 | We then add ATP to 1 mM, nucelotides to 0.5 mM, four units of T4 ligase, and 2 | ||
units T4 DNA polymerase. This reaction is done in PE2 buffer (10X PE2 200 mM | units T4 DNA polymerase. This reaction is done in PE2 buffer (10X PE2 200 mM | ||
| − | Tris Ph 7.5, 100 mM | + | Tris Ph 7.5, 100 mM MgCl<sub>2</sub>, 100 mM DTT). Total volume of the reaction is 20 |
micro liters. | micro liters. | ||
Revision as of 10:55, 18 August 2009
COMPARE to my protocol
Primer phosphorylation with T4 PNK: we phosphorylize 200 pmol of primer in 2ul reaction mixture
Annealing: 10 pmol primer, 0.5 p mole ssDNA in 10ul PE1 buffer (10XPE1: 200mM Tris ph &.5; 100mM MgCl2, 200 mM NaCl, 10 mM DTT).
phiX174 DNA has a great deal of secondary structure. To anneal, we heat some water to boiling, remove from heat, place the tube in the water, refrigerate and let it cool to 4C, takes about an hour or two.
We then add ATP to 1 mM, nucelotides to 0.5 mM, four units of T4 ligase, and 2 units T4 DNA polymerase. This reaction is done in PE2 buffer (10X PE2 200 mM Tris Ph 7.5, 100 mM MgCl2, 100 mM DTT). Total volume of the reaction is 20 micro liters.
Keep on ice for 5 minutes, then room temp for five minutes, then 2 hours at 37C. After wards the reactiosn can be frozen. We typically dilute the reaction 1/10 before transformation. The transformation mixture contains 100 microliter competent cells, and we use 2 microliter of the diluted synthesis reaction.
We have found that CaCl2 treatement gives us the best transformations. We also get much better efficiencies if we make the competent cells the day before, and refrigerate them overnight.
We incubate the transformation mixtures for 30 minutes on ice, heat shock for one minute at 42, then ice again for 5 minutes, and then we plate. This typically gives us 200 plaques per plate.
The repair can go either way in the cells, so you will need a way to either directly select for your mutant, or an efficient way to screen the plaques by toothpicking into indicator lawns.
