I'm doing transcription mapping of k. lactis tRNA nucleotidyltransferase. I isolated total RNA, and used RLM-RACE to generate cDNAs of the transcripts. These cDNAs contain A-overhangs because they were generated using Taq polymerase. I captured the cDNA transcripts using a T-tailed pBS that I generated by digesting pBS with EcoRV and then adding Taq in the presence of T nucleotides. Then I ligate the T-tailed pBS with the A-overhang cDNAs to generate my clones and do pick white colonies on plates with X-gal and IPTG.
I've been having a lot of trouble with my ligations (I get very few blues and whites) and even more trouble in screening with RE digests. Essentially I thought that I wasn't getting any digestion, and blamed the miniprep protocol. I've addressed this problem in the "best miniprep" thread and many members there were helpful but today I went to the lab and had a talk with my supervisor.
So I went to the lab today and talked with my supervisor and another research scientist in the lab. I showed them my "undigested" plasmid, and then told me that it was digested, but was only linearized. Oops. I'm still kind of new to this. Anyways, the problem now seems to be that my vector, which normally has 2 pvuII sites (and so gives a nice band of about 400 bp without insert), only has 1. Taken together with some previous data I collected when I accidnetly left a digest overnight and found that all of my pBS-pvuII control was completely chewed up (nucleases), a colleague suggested that my vector was also chewed up (since the vector I'm using is derived from the same pBS that was chewed up by nucleases). So when I made my T-tailed vector, a lot of it was probably chewed up by this nuclease, and when I go to do a RE digest on it, I only see linearized vector and no insert band.
This probably is why I'm not getting the digestion pattern (a linearized vector and a 400+ bp fragment) that I've expected. Although I'm not sure how this would affect my ligation problems (but I'll get into that again if I still have problems).
I wanted to know what you guys think of this and want to keep this thread open (for myself), so I can come back here to report on any further difficulties I'm having. I'm an undergraduate and I'm loving this stuff, but it can be frustrating! Especially when I now realize that I have to start over!
SO tomorrow I'm going to extract pBS from a blue colony that I picked off a plate, do GeneJet on it, treat it 2x with phenol, and 2x with ether, then precipitate it. THen I'll set it up for digestion with EcoRV and go on with T-tailing from there.
TA cloning project
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