BISC209/S11: Corrections to Protocols in 2011

ZeroBlunt® TOPO PCR Cloning Kit

Once again bad news and good. It has not been an easy semester for molecular biology in microbiology lab. I still don't know why your pcr amplifications of 16srRNA didn't work initially, but we overcame that with some concentration of the DNA and adjustment of reagents. Sadly, the cloning and transformation that you did on Wed. didn't work either. Sigh. I do, however, think I know why that didn't work and we have fixed it. Although we had evidence of good amplification of 16s rDNA in the pcr redo (as you saw in the gel image posted), the gel was run before we cleaned up the small DNA fragments (dNPTs, primers,degradation products). I think we lost a lot of DNA during that purification process. I nanodropped your pcr products on Thurs after I recovered from my horror that we had NO transformants on any of our plates and discovered there wasn't much DNA there, which may have been part of the problem. We concentrated DNA by combining what was left of all the D products, E products, etc. and evaporated off the excess water in a Speed-Vac for an hour. That concentrated our DNA to about 20-40ng/microliter (rather than 0-8!) Then we re-did the cloning and transformation on one concentrated D, E, and F sample using the same topoisomerase kit that we used and did another run at the same time using a slightly different enzyme (a ligase) and vector and found out this morning when the ligase worked and the topo didn't, that the kit we used on Wed. had a crucial reagent that was bad. The short answer is that no transformants was the fault of a bum topo cloning kit. Needless to say, Invitrogen will be hearing from us! What this means for you, is that all is well and we can continue our culture-independent identification of bacteria in your soil community. We have clones to grow up in a deep well plate next week and then to make glycerol stocks to send away for sequence analysis. All is well.

Cautionary note: DO NOT report all the issues and redos in this process in your M&M!!! M&M is not a description of your lab days and everything you did to accomplish your goal. It is a description of how each goal was reached. If you want to change the kit information in your protocol that describes the separation and amplification of E coli clones with your 16srRNA soil bacterial gene inserts, that is appropriate. I am attaching the kit we actually used to this announcment. You need not change the transformation description since I used the same E. coli cells that you did on Wed. and the selection with kanamycin, ccdB and lacZ regulation is the same. You need not change your description of the pcr amplication of 16srDNA or the clean up since we just repeated the same protocols and there is NO need to report that you did it twice to get it to work. Link to pdf: http://products.invitrogen.com/ivgn/product/K270020?ICID=search-product#manuals

Description of K270040 version of kit for $768 The Zero Blunt® PCR Cloning Kit offers an easy method for high-efficiency (>=95%) cloning of blunt-end PCR products amplified with proofreading thermostable DNA polymerases. The Zero Blunt® PCR Cloning Kit uses the multipurpose cloning vector pCR®-Blunt and a one-hour ligation step. The vector features:

   * ccdB gene for positive selection (1)

R I sites flanking the PCR product insertion site for excision of inserts

   * Choice of kanamycin or Zeocin™ resistance for flexible antibiotic selection

   * T7 promoter/primer site for in vitro RNA transcription and sequencing

   * M13 forward and reverse primer sites for sequencing or PCR screening

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12