I am currently trying to prepare a library for illumine deep sequening using the yeast Candida glabrata. Ive been having difficulty generating a high quality library. My collaborators believe that it is my genomic extraction that is causing the problem, with some protein or some leftover buffer from a previous step. I was using a modified version of the master pure yeast DNA extraction kit with a phenol chlor extraction at the end. Whenever I would go to sonicate my DNA I would have a large amount of fragments that were of low MW (less than 50 bp according to the BioA) a low amount of normal fragmentation (between 250 and 400 bp) and a large amount above 700 or so bp, with the lower than 50 bp outweighing the others by far in terms of molarity and weight. As such they recommended that I use a new extraction protocol and do ti alongside someone in their lab.
Based on their recommendation I am currently trying the ZR fungal/bacterial genomic mini prep kit. Me and another ran the protocol using a highly saturated glycerol stock. Their prep had great fragmentation mine looked like a lot of protein contamination. When i nano dropped the samples the one that worked had a 260/280 of 2.0 and a 260/230 of .7. The one that did not work had a 260/280 of 1.6 and a 260/230 of .35
Based on the fragmentation (too much in higher MW fragments) and the low 260/280 I believe that the fragmentation for the second one failed due to protein contamination.
So i started taking to zymo and they gave me some recommendations. After following them I got samples with a 260/280 of 1.85 and a 260/230 of .~1.9-2.0. So based solely on the nano drop these samples seem much better. (they also have a higher yield).
However when I run them on a gel I get a very concerning smear. The first gel image includes the original attempt, in which one had good fragmentation the other bad fragmentation. It is a 1kb NEB ladder. The third well with sample was the extraction that had good fragmentation, the fifth well with sample had the bad fragmentation. The second image contains my most recent attempts with all but the first 2 sample containing wells coming from the extraction kit. It looks like there are fragments in this smear from 10kb down to 1kb if not lower. However if I am looking at the first one correctly it seems that the one that had good fragmentation also had a smear that went to low MW. It may just not be as visible due to the large difference in initial loading concentrations. My question to everyone is whether or not they think any of the 4 samples in the second gel image would be acceptable to carry over for fragmentation and subsequent library preparation?
Thank you for your time
Hello Mat,
Thank you for responding so quickly. In regards to the usage of the master pure kit I am following a protocol given to me by a sequencing core for what they use to purify DNA for sequencing. In it they run through the master pure kit then add an additional RNase A step followed by Proteinase K incubation, which the phenol chlor is used to remove them.
So even though it seems that the original extraction using the zymo kit had good fragmentation and looked like it also had degradation you believe that the new ones would not work well for sanitation?
Andrew
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