Since last summer plasmids sequencing always fails in my lab. We use Zyppy miniprep kit. The other lab using qiagen miniprep kit never have problems. Now we have to PCR our fragment first, and then run DNA gel electrophoresis to cut the gel slice or else we cannot get reading at all. I am so confused about Zyppy miniprep kit. Since it gives a good reading of A260/A280, A260/A230. However, sequencing just don't work. Even for the clones worked before. BTW, We both send our samples to Eurofins Genomics.
What's the concentration of your plasmid after doing the miniprep? We use a local company (Eton bio) which requires our concentration be between 75-125 ng/uL and we send in 10uL at a time.
We increased the amount of cells we use with the kit. We grow 3mL cultures overnight, spin down the cells and resuspend in 600uL of water before continuing on with the lysis and other steps.
Are the primers ok for sequencing? Has this happened with multiple primers?
Thanks for your answer. Our plasmid concentration is normal, 100ng/uL-300ng/uL. We treat the cells in the same way as you do. The primers work if we PCR out target fragment. But if we directly use it, it does not work
Could there be some sort of inhibitor in one of the reagents of the kit? If you can get sequencing the work after PCR and gel electrophoresis, then that could mean there was something in the reagents that you are getting rid of. Have you tried a clean and concentrator kit?
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