Dear all,

I want to delete PEX11 gene in a Sacchromyces cerevisiae deletion strain. I have already Sc PEX11 deletion strain which is indicated in this article, but don't have any plasmids having the deletion cassette. So, I will amplify Pex11 deletion cassette from the genome of this strain by cPCR. Then I'll transform it into the strain of desire. For colony PCR and southern blots, I should have the sequence of this deletion cassette (i.e plasmid map).

I've looked at the Materials and Methods, and followed how they constructed the plasmids they indicated.. But, I am not able to make the maps as they indicated..

They wrote that: " Plasmid p27-1, containing a 3.8-kb EcoRI fragment, was isolated from the subgenomic library. A 2.2-kb ClaI/SpeI fragment of p27-1, which correspond to the entire PMP27 gene plus 5' and 3' noncoding regions, was subcloned into pBluescript SK+, resulting in p27-10." 

Based on this info, I made p27-10 (5137 bp). In the section where they explain deletion of PMP27 (PEX11), it is written: 537 bp XbaI-PstI fragment of p27-10. However, when I check it on CloneManager software, it shows 496 bp fragment.. 

It is possible that it is written wrong in the article.. But I want to be sure about it.

My second problem is with making plasmid pJJ282 (pJJ250) from which LEU2 gene was cloned into p27-10. In the article from Jones and Parkash (1990), they wrote that LEU2 gene was isolated from YEp13 plasmid on a SalI-HpaI fragment, and cloned into pUC18. However, pUC18 doesn't have HpaI restriction site. Then, how did they clone it? I couldn't understand..

I hope you could help me with this issue.

Thanks in advance,

Arman

http://jcb.rupress.org/content/128/4/509.long

http://onlinelibrary.wiley.com/doi/10.1002/yea.320060502/epdf

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