Hi everyone. I am currently using Invitrogen pCR-Blunt II-TOPO kit to ligate my PCR fragments into the TOPO vector. However, most of the time I obtained very few E.coli colonies (fewer than 3 on average) on the Kanamycin plate (the TOPO vector is Kan-resistant). For the colonies that I pick, the chances are the insertion fragments are not consistent with the lengths of my PCR fragments based on EcoRI digestion (thus not my PCR products). My PCR fragments are mostly 1kb-4kb, so the length shouldn't be an issue (I even got 20 colonies once for a PCR fragment of >5kb). Sometimes out of curiosity, I sequenced some of the minipreps from the colonies, and it turned out the insertions were random E.coli chromosome fragments.
I have tested and changed as many reagents as possible that are involved in this process. Moreover, I have no problem with other experiments such as T4 ligation where techniques like minipreping, gel extraction, transformation etc. are also used. My PCR technique in isolation also seems OK since I mostly get a single bright band on the gel. I have looked at the official website of the TOPO product, but none of their advice worked in my case. Other people in my lab seem to be quite happy with TOPO cloning so it shouldn't be the kit that is contaminated (besides, we have changed many TOPO kits over time but my problem is persistent). Thus, I believe there is some part in the process where I can easily make mistakes but I just haven't noticed yet. Has anyone had similar experiences and hopefully can share some suggestions? Thanks!
Hi Jian,
Did you manage to solve your problem?
One thing you didn't mention in your question was what polymerase you are using.
Just in case you were unaware, since you are using a blunt TOPO kit you should not generate your PCR product with Taq polymerase, which will add an untemplated 3' A overhang that will interfere with the blunt TOPO ligation.
Instead you should use a proofreading polymerase (i.e. NEB's Phusion), which does not add untemplated 3' A overhangs to products. Alternatively, if you use Taq, these 3' A overhangs can be removed by incubation with Klenow fragment.
If your previous inserts turned out to be fragments of bacterial chromosome, I suggest performing your PCR, running on a gel and band extracting the band of interest, then re-perfoming PCR using this extracted band as input DNAand band extracting again. This should dilute out any chromosomal DNA contamination.
Good luck,
Chris
Christopher Roberts Hi Chris, thank you so much for your suggestions.
Yes, I always use Phusion polymerase from NEB and this combination used to work perfectly for me. Just since a point everything started to become so temperamental. And now I still constantly have this problem where TOPO gives no colonies at all.
Yes I also tested my gel extraction products by running them on the gel. And there was no problem with their purity or other quality issues. However, thanks for your advice!
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