I need to subclone a gene into an unusual vector that has only EcoRI at the insertion site. I don't have a construct in which my gene is flanked by EcoRI sites, so I will have to PCR amplify it to add them no matter what. My gene also has an internal EcoRI site. So, instead of doing a partial digest followed by non-directional cloning, this seems like a great opportunity to try Gibson Assembly. I was thinking that I could digest the vector with EcoRI and generate my insert by PCR with primers adding 30 bp of vector sequence on each side. My question is, won't the vector anneal to itself and reclose at a high frequency? Perhaps the reaction temperature will be too high for a small overlap to anneal and the insert will be favored? I know the other approach is to amplify the entire vector to create a blunt insertion site, but I'm worried about introducing errors. If anyone has any experience with this type of situation, I would appreciate any advice. Thanks!
Hi Micheal, sorry for the delay I have just come across this question whilst searching for something else. You probably have solved your problem by now, but if not I have added some suggestions below.
Your idea of gibson assembly is a good one, you will not get excessive religation, as the T5 exonuclease in the gibson reaction will remove the ecoR1 overhangs probably before the ligase has a chance to act. Just be sure to design your homology arms ignoring the overhangs on the Eco site, as 5' overhangs will be chewed off. Be careful if there are other restriction sites nearby as gibson arms are not too keen on lots of palendromes, but I am assuming there are not or else you would not be limited to ecor1
Alternatively just clone a cassette into your vector with the restriction sites of choice for your insert, by annealing two oligonucleotides together. Screen some clones and in theory you will get half in each orientation. That way you can choose the clone you want and you have a more useful vector for the future. This of course assumes that you can add a few bases of DNA to your vector, without disturbing its other properties. Without knowing what your vector is it is difficult to tell
Hope this helps
David
Hi Michael,
As an alternative to using Gibson Assembly, you could use compatible cohesive ends to clone your gene into the vector and avoid the internal EcoRI site in your gene. If you put Mfe I sites on the end of your gene you can ligate them to the EcoRI sites in your vector. You would still have the problem of the gene being ligated into the vector non-directionally though. Hope this is helpful.
Good Luck!
It is not advisable to linearised plasmid by single RE, as the chance of reaneal is high. To prevent re-anealing, you should use 2 REs (which the ends are not complimentary) OR if you are using only single RE, dephosphorylate the plasmid with alkaline phosphatase (like CIP, CIAP, TSAP, rSAP) to minimise self-annealing of the plasmid.
Blunt ends annealing is considered to have lower efficiency of annealing that sticky ends. Products from the non-directional ligation could be filter out easily with PCR (1 vector targeted plasmid + 1 insert targeted plasmid). You should have plenty of clones available for screening.
With DNA sequencing analysis, you can confirm if your whole construct is correct.
If you are only aim to insert a single fragment, and the overall final recombinant plasmid won't be too large, you may want to consider overlapping extension PCR (http://www.rf-cloning.org). This is easy to design and works well. You no need to consider about the RE sites.
All you need is high fidelity polymerase with higher fidelity (like Fusion polymerase, Q5 polymerase, etc., not pfu). And also DpnI RE. The drawbacks of this technique are (1) you might need to screen 100-200 clones for some cases, (2) the amplification is linear, so the transformation rate is low too (works on lab prepare Cacl2 cells), (3) size of the overall plasmid should not be too large for the polymerase amplification (~10kb will still be acceptable).
Good luck
Hi Micheal, sorry for the delay I have just come across this question whilst searching for something else. You probably have solved your problem by now, but if not I have added some suggestions below.
Your idea of gibson assembly is a good one, you will not get excessive religation, as the T5 exonuclease in the gibson reaction will remove the ecoR1 overhangs probably before the ligase has a chance to act. Just be sure to design your homology arms ignoring the overhangs on the Eco site, as 5' overhangs will be chewed off. Be careful if there are other restriction sites nearby as gibson arms are not too keen on lots of palendromes, but I am assuming there are not or else you would not be limited to ecor1
Alternatively just clone a cassette into your vector with the restriction sites of choice for your insert, by annealing two oligonucleotides together. Screen some clones and in theory you will get half in each orientation. That way you can choose the clone you want and you have a more useful vector for the future. This of course assumes that you can add a few bases of DNA to your vector, without disturbing its other properties. Without knowing what your vector is it is difficult to tell
Hope this helps
David
Hi David. I ended up doing PCR and adding MfeI sites as Tom suggested above. Luckily it was a small insert, so I didn't find any errors in the sequence. I definitely want try Gibson assembly at some point, but it sounds like it takes a bit more expertise to design than it appears from the NEB website. In the past, I have had good luck with inserting annealed oliqos between two restriction sites, but the one time I tried inserting into a single site, the oligo formed varied multimers Thanks very much for your advice!
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