The gene I am working with is toxic to bacteria and frequently causes random deletions/insertions when cloning in E.coli. Therefore I have cloned my construct directly into yeast (my expression system). I have done a small scale screen for the expression of my protein which suggests I have transformants. However, I now want to confirm this by sequencing. Can anyone suggest a plasmid extraction protocol which yields DNA of a quantity/purity suitable for sequencing. My gene is quite big so I need a relatively high yield. Most of the kits I have come across so far only mention up stream use in PCR or bacterial cloning. I recently used a kit from Thermo, which gave a very high ratio of genomic DNA relative to plasmid. Any suggestions?
Hi Eleanor,
What type of yeast/strain are you using?
Plasmid extraction in yeast typically yields very low amounts and the quality too poor for sequencing. Therefore after extraction you typically transform into bacteria to amplify the plasmid and extract again to get good clean DNA for sequencing. But since your gene is toxic to bacteria that does not sound like a viable option. The alternative is to PCR amplify your gene after plasmid extraction from yeast and then directly sequence the PCR product.
But can't you prevent expression in E. coli??? There are several promoters in E. coli that can be down regulated or repressed. That would be an ideal system to amplify plasmid without the deleterious effects of the gene product.
Thanks for your response Tom. I am using FGY217 S.cerevisiae. PCR amplifying and sequencing the PCR products was my original strategy, but I am currently having a little bit of difficulty with it. It was also recently suggested to me that by only having amplified PCR products sequenced I am adding another degree of removal between the sequence I obtain and the actual sequence in the yeast, and that since I have a lot rested on the sequence being right (structural studies), I should really try and sequence the actual yeast extracted plasmid. So I just was wondering if this is at all possible. Sounds like perhaps not so I will persevere with the original plan! Thanks again!
Hi Aaashiq, yes theoretically the protein should not be being expressed in E.coli at all but I've always had problems with it (maybe there are cyptic promoter sites somewhere on the plasmid). I've tried Able K e.coli cells which should reduce the effective copy number and therefore toxicity, and SURE2 which are meant to prevent unwanted re-arrangements, reduced growth temperatures, only growing on solid media, and really nothing seems to help! It's a membrane protein which is well documented to be toxic in e.coli. So I am quite reluctant to put it in e.coli, I've really wasted a lot of time with it before!
Hi Eleanor,
Just use a good high fidelity polymerase for your PCR. You can also run several independent PCR reactions and get them sequenced. If the same mutation appears in the sequencing runs then it is probably in your plasmid template. If you see different mutations (random) in the sequencing runs then that is probably resulting from your polymerase.
Good Luck!
Google shows many kits and papers for isolation of pDNA directly from yeasts. For example, http://www.ncbi.nlm.nih.gov/pubmed/12137773. Mostly, they are all varieties of the good old alkaline lysis method. The yields are low, so you need a high amount of culture.
To improve the quality of pDNA after the isopropanol precipitation or commercial columns, you can try spermidine precipitation (1.5-5 mM in TE) (http://www.nature.com/nbt/journal/v17/n8/full/nbt0899_822.html). Usually it gives DNA of high quality.
We extensively tested both single copy CEN/ARS and 2 µ yeast plasmid constructs for the paper mentioned by Misha (http://www.ncbi.nlm.nih.gov/pubmed/12137773). The copy number depends on the gene product's toxicity/neutrality in yeast. In our case, TAF1 of yeast was showing same (~1 copy per cell) for both CEN/ARS and 2µ plasmids. You also have to consider that it is in stark contrast to E. coli, which may harbor 200 copies of a plasmid. If you are seeing low yield, it is not surprising. However, I will strongly recommend that instead of scaling up the amount of yeast in one column, you try multiple column approach following that protocol we published. It can be used with almost any commercially available bacterial plasmid kit (after initial lysis by using lyticase enzyme). It gives high quality plasmid. But you have to remember that yeast has RNA plasmid that does not get easily digested by regular RNase treatment. Hope it helps. Good luck.
Thanks Misha and Madhu for the protocol, I will give that method a try.
Hello Madhu, I tried the plasmid extraction method in your paper. I think I can see a band corresponding to my plasmid, however there is a very smeared appearance. I think it may be due to DNA degradation (rather than protein) as the 260/280 and 260/230 are good (1.87 and 2.08 respectively). Did you ever experience such problems?
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