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| Yeast Forum Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species. |
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| I am a new graduate student rotating in a yeast lab and I am having trouble amplifying the James genomic library (C1,2,&3) for a 2-hybrid screening using GAL4 as the construct. I have very old samples of the plasmid DNA as well as new libraries in E. coli. I have tried amplifying the libraries (from the old plasmid DNA) using TOP10 electrocompetent E. coli and cannot get a high enough transformation efficiency. Thus, with new libraries (in E. coli) I am trying to fomulate a protocol for amplifying the 5 x 10^6 clones properly and isolating the plasmids (at least 100-500 ug DNA). Thus far, using the QIAGEN HiSpeed Midi-Prep Kit I have only been able to extract 5 ng/ul plasmid. Is there any protocol for amplification as well as hints/suggestions for getting higher yields on large-scale plasmid preps? Thanks in advance for any help! G |
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| amplification , hybrid , isolation , library , plasmid , problems |
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