Dear all,
I am cloning N-terminal GFP with Linker-MCS exchanging with C-terminal GFP in PBCAG-eGFP (addgene 40973) vector, the enzymes I used are EcoRI/NotI. This sounds like a very simple cloning, but the fact is, I failed to get colonies with GFP PCR products. Then I cloned the N-GFP PCR into TOPO vector, this worked well. However, again, I failed to get colonies with the insert digested from TOPO plasmid!!
I also only digest the PBCAG-eGFP (addgene 40973) with EcoRI and religate this with the Quick ligation kit we used and transform into the XL10-Gold bacteria. I got plenty of colonies with this, which suggests that the vector with GFP insert is not toxic for the XL10-Gold, and the ligase I used works.
I am now really helpless, have no idea where could the problem be and what should I change.
I added the protocol and one gel with ligation here, I think the ligation works, because the band is higher than digested vector.
I appreciated your suggestions!
Dear Shu,
it is hard to say. Your approach seems to be very straightforward. If you try again, I would suggest to use the the fragment from the TOPO vector and check anliquot of the fragment on a gel. Also get the sequence analysis of the Topo insert, and specially of NotI and EcoRI site. The only thing, I can imagine is that your isolated fragment is primarily the result of one restriction enzyme, e.g. your EcoRI site and an EcoRI site in the Topo polylinker,
Best,
Uwe
Dear Uwe,
your suppose is brilliant, in the TOPO vector there are EcoRI in both sites, and also NotI in 3´-site. But under normal digestion conditions with 1µl HF enzymes and 1h 37°C, all sites should be digested. The TOPO sequence analysis has been done, and it contains the correct inserts plus the restriction sites.
To avoid the EcoRI fragment, I can first cut with NotI for 1h, then add EcoRI for another 1h.
Best
Shu
Do a PCR on your ligation reaction if that is positive for what you want, then maybe you can switch the restriction site, for example amplify across the ligation junction from your ligation reaction plus upstream and downstream in the vector e.g include the xbaI site from the chimeric intron down to the bglII site then digest the vector and insert with those and try again.
Hi Hanna,
did you tried this for your cloning and it worked? Because I dont know what is the difference between this strategy and my old one. If I can successfully get PCR with my ligation, mean ligation worked. Then the question is why there grow no colonies. Will the new digestion with other enzyme and new ligation help? Because the new ligation will give me the same product.
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