I want to assemble 1 fragment of 771 bps in vector backbone of about 7kb which is digested with HIndIII. I got colonies upon transformation but none of them have the insert. What could be the problem? I did a PCR on my insert and then did a column purification on it. I did a column purification even on my vector backbone prior to setting up the gibson assembly reaction. I can't figure out what is the problem
This doesn't normally work in this way. You have too much uncut vector that escaped the HindIII. It's too little to see on a gel, but enough to swamp your Gibson assembly. You should either make the vector by PCR using a long range polymerase, at the greatest dilution that will still amplify. Or if you must cut the vector, gel-purify it, to remove the (invisible) uncut vector bands (supercolied & relaxed forms).
Hi Pooja,
I have done recently a similar gibson cloning, with a BB digested with a single enzyme. Here's couple things you can double check.
1) Are you digesting your BB long enough? As John mentioned, cells are sensitive to small concentration plasmids. - Digest your BB with HindIII for couple hours, specially if you are not using a HF-enzyme. You really want to ensure that you don't have any uncut vector left. Most people will run it on a gel as well. That's probably smarter to do.I have done quite long digest (3-4 hours) and then directly PCR purified and was fine too. Just to be safe, run it on a gel, cut the bands, gel extract and PCR purify.
2) Your insert: Ensure that you have designed your primers correctly (the bp annealing to the backbone), it is the 2nd reason why it is not getting ligated into it after uncut BB contamination. DpnI your insert as recommended.
3) Ligation/transformation: the gibson gives very specific instruction on those, just make sure you are following them (e.g.depending on cells/type of transformation, you need to dilute your ligation, ratio of BB vs insert, total amount of DNA for the ligation...)
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