Problems experienced from adding 3xHA epitope to the C- terminus of some interes - Problems experienced from adding 3xHA epitope to the C- terminus of so (Aug/22/2007 )

Problems experienced from adding 3xHA epitope to the C- terminus of some interested proteins
Method:
Sequentially introduced ~400bp DNA fragments corresponding to the upstream and downstream sequences from the stop codon of our interested gene to the 3xHA cassette derived from Mark Longtine’s original construct. Then PCR amplify the recombination cassette. After checking the PCR products with electrophoresis to verify the size, the PCR products were cleaned up and recovered for yeast transformation by electroporation. The amount of recovered DNA was estimated by running an aliquot with agarose electrophoresis.

Problems encountered in this experiment.

First, after transformation, colony PCR verification on those colonies grown on selective plates frequently gave no result, occasionally with faint band at the expected size suggesting inclusion of the tag to the target gene. However, there were also cases when the PCR products corresponding to the size of DNA fragment with no insertion of the cassette.

Second, after streaking the glycerol stock which has been previously verified by colony PCR two months ago from -80℃ on selective plate for single colony or even using the glycerol stock to repeat the colony PCR with the same condition as used before, I can not get the right PCR product this time. Instead, the PCR products correspond to the size of DNA fragment with no insertion of tag, although all of them can grow on selective medium.

The above problems have puzzled me in the last several months and greatly hindered the progress of my work. I am very grateful to anyone who can provide some suggestions and answers to help me cope with these obstacles.

I apologise but I am still not entirely sure about the exact problem.

My suggestion would be to try to sequence the plasmid you have or atleast try to digest it to check for the right sized fragments. This would be better than colony PCR for DNA verification.

first,thanks for your suggestion,a good idea.but i think it is difficult for me,because i want to verify the cassette has inserted to the genomic DNA by colony PCR.