MSH2 project problems in BIO125, Spring 2013

In BIO125 Spring of 2013, we had three major problems:

1) The 5FOA assays gave opposite results 
 Functional assay of MSH2 and its mutants on 5FOA plates gave results that were opposite to expectations: Wildtype MSH2 transformants gave the largest number of colonies, pRS413 vector control (msh2Delta) gave the least number of colonies. The yeast strains for these assays were the pre-transformed yeast strains shipped directly from our collaborator, partly because students' transformation did not use -URA selection for the reporter marker, partly because we thought the pre-transformed yeast should work. It seems that the wildtype and vector control (msh2Delta) were somehow mixed up. 

Cell concentrations in the three samples are based on OD600. All three strains were grown only overnight, and their OD values are so low that we had to spin them down and concentrate them. We then diluted the samples to similar OD600 and spotted them on 5FOA plates. Ideally, the wildtype MSH2 should give the least number of colonies on 5FOA, because its low mutation rate in the micro-satellite region of the URA-reporter gene. The vector control should have the higher number of colonies than the wildtype MSH2, but unfortunately not.

Possible ways to address this problem: 
Redo transformation with the proper -URA selection.

2014 Feb 10: URA3-reporter is under LEU promoter that are activated in -LEU-Thr conditions.


2) Reporter strain  showed the same band with wildtype MSH2 by PCR 
Dr. Steve Kiokio performed PCR using our MSH2 primers, and saw the expected bands in the reporter strain. It is possible that MSH2 mutation in the reporter strain is not a full-gene deletion, and our primers simply amplified the region still remained in the truncated MSH2 on the chromosome. 

Possible ways to address this problem: Redesign PCR primers, seek advice from original authors.
The primers  BIO125 used to amplify a 845bp fragment of MSH2 in the coding region are:
MSH2FOR 1395 5' CAA ATT CGA AGA AAT GGT TG 3' 1414
                            or CAAATTCGAAGAAATGGTTG
MSH2REV 2239 5' CAA CCA TAA ATG TGG AAA CAC C 3'
                            or  CAACCATAAATGTGGAAACACC
This pair primer has very low annealing temperature, and gave non-specific bands.
Our laboratory instructor, Dr. Kioko extended these primer to raise their annealing temperatures, but he obtained the same results.
The new primers are:
MSH2F 5'T GTC CAA ATT CGA AGA AAT GGT TG3'
          or  TGTCCAAATTCGAAGAAATGGTTG
MSH2R 5'T CAA CCA TAA ATG TGG AAA CAC C3'
          or  TCAACCATAAATGTGGAAACACC


Possible ways to address this problem: 
Contact the author for their PCR primers and ask for MSH2 deletion details. 

2014 Feb, endogenous MSH2 was disabled by disrupting of its promoter.


3) Western blot of total protein isolation did not work. 
 Pull-down of whole cell lysate  by HA antibody did not show any bands in western-blot. We suspect that MSH2 protein are trapped in the nuclear in the lysate, and may be thrown mostly thrown away in the precipitated portion.

Possible ways to address this problem:
Revise protein isolation protocol. Seek advice from the original authors.