I'm currently trying to capture a biosynthetic gene cluster using Transformation-Associated Recombination (TAR) in yeast. After identifying positive yeast clones, I extract, via an alkaline lysis method, the plasmid from yeast and electroporate it into E. coli DH10B.
However, I have not been able to find any positive hits upon cPCR on Eci clones despite testing about 100 colonies using the same diagnostic primers I used to identify the yeast positive clones. Then, on some of the E coli that I pick and miniprep, I consistently only see my original capture vector (with no gene cluster inserted).
The issue may lie in the yeast plasmid extraction, the transformation, or the plasmid isolation prep from E coli. I know that yeast plasmid extractions are hardly ever clean and tend to be "dirty," contaminated with yeast gDNA and other DNA it has inside due to the IPA precipitation required to perform the method. That tends to lead to poor transformation efficiency into E coli. But I feel like I'm stuck going in circles trying to bring the plasmid into E coli and isolating it. if anyone has ever worked with TAR and has experienced any troubleshooting at this stage of the process, I would appreciate insight. Or any advice on things I can try to better improve yeast plasmid extraction or transformation/isolation in E coli. Many thanks!
Transforming and isolating a large plasmid in E. coli can sometimes be challenging, but there are several troubleshooting steps you can take to optimize your protocol:
By carefully optimizing each step of the transformation and isolation process and troubleshooting any issues that arise, you can increase your chances of successfully transforming and isolating large plasmids in E. coli.
Hi this a problem I constantly had before. You are right the main issue is the plasmid extraction from yeast. One solution that worked consistently in my hands for this, is to use Zymoprep Yeast Plasmid Miniprep II (https://zymoresearch.eu/collections/zymoprep-yeast-plasmid-miniprep/products/zymoprep-yeast-plasmid-miniprep-ii) and transform in commercially prepared competent cells that are used for genomic library prep (ex NEB10 beta high-efficiency, chemical competent).
Hanna Alalam I really appreciate your response. I think the issue with using the newer zymo kits is the size of my plasmid... My plasmid is 44 kb, but the zymo kit is rated for 23 kb plasmids. The Miniprep I kit has no size limit because it is an IPA precipitation that will pull down DNA of all sizes.
Ok I see. in that case you could for another alternative, you could try using protocols used for pacbio sequencing, this way you will get high molecular weight DNA that far exceeds the size of the plasmid then you transform that in coli (You do not need to worry about the presence of the yeast DNA it will not interfere that much with the transformation).
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