1. Prepare 1M Hydroxylamine solution.
- 0.35g hydroxylamine hydrochloride (H3NO-HCl)
- 450 µl 5M NaOH
- 4.55 ml ice cold sterile MQ water
Make sure pH is around 6.7 and keep solution on ice
Incubate 10 µg plasmid DNA with 500 µl hydroxylamine solution for 20 h at 37 °C in an eppendorf tube. Seal eppendorf tube with parafilm.
Purify DNA from hydroxylamine using Qiagen column…(Purification of plasmid DNA prepared by other methods…page 31 of 7/99 QIAprep Miniprep Handbood)
- add five volumes of Buffer PB to one volume of DNA and mix
- load column (0.75 mls at a time) and centrifuge 1 min at max speed
- wash column with 0.75 mls Buffer PE (stand 1 min, centrifuge 1 min at max speed)
- centrifuge one more time (1 -3 minutes at max speed)
- elute DNA with TE or EB (50 µl), stand 1 min, centrifuge into new tube)
Check recovery of DNA from mutagenesis by running on agarose gel and transform into appropriate yeast strain. Use approximately 500 ng to 1 µg DNA per transformation.
Forsburg Lab anecdotes:
We used a plasmid shuffle strategy for mcm7. We mutagenized from 16 to 22.5 h at 36 *C or 1 h at 70 *C, transforming by electroporation with 5 µl mutagenized DNA per electroporation. We screened about 10,000 transformants and recovered 3 ts mutants (all mutagenized for 22.5 h at 36*C). Two contained identical mutations. One of the mutants did not support growth when reintegrated into the genome in single copy. The other is a well behaved, tight ts.