Hi,
I double-digested a plasmid to obtain a 2.5kb DNA fragment that I want to insert into a larger plasmid (11.7 kb). However, I failed to see that my double digest also yields a 250bp fragment with the same restriction sites. Now, both are in the ligation reaction, and I am wondering whether the 250bp fragment will completely outcompete the 2.5kb fragment, or basically what the chances are of getting the 2.5kb fragment inserted despite the 250bp fragment's presence in the ligation reaction.
I ran a diagnostic gel of cut vs. uncut plasmid, and the 250bp band is visible, however much weaker than the 2.5kb band; but this is probably to be expected from the size differences.
Thanks,
Patrick
I think that you should run an agarose gel and purify the large fragment. The small fragment is equimolar with the large but being smaller I think that the ends of the fragment are more likely to be on the outside of the molecule not hidden in the folds of the structure which seems more likely with a large fragment so I think that you will have more transformants of the small. Also the presence of 2 fragments will mean more insert to insert ligations which will lower the efficiency of the wanted ligation both by removing insert and blocking one side of the insertion site.
I think that you should run an agarose gel and purify the large fragment. The small fragment is equimolar with the large but being smaller I think that the ends of the fragment are more likely to be on the outside of the molecule not hidden in the folds of the structure which seems more likely with a large fragment so I think that you will have more transformants of the small. Also the presence of 2 fragments will mean more insert to insert ligations which will lower the efficiency of the wanted ligation both by removing insert and blocking one side of the insertion site.
Thank you for your answer, Paul. I agree that cutting the 2.5kb fragment from the gel is the best option, and will do that next time. About the insert:vector molar ratio, I used 7:1 for the 2.5kb insert, which actually makes it 14:1, calculating with both possible inserts, and assuming equimolarity (which may actually not be the case, I think, because I hope that column purification favoured the 2.5kb insert over the 250bp one.. however, judging from the gel, it seems roughly equimolar..).
Hi Patrick,
You definitively need to gel purify your 2.5kb fragment otherwise you will never clone it into your vector.Even though columns may favor fragments larger than 250bp, such a small fragment will always out-compete the larger when cloning. If you wanted to clone the smaller fragment, removing the bigger fragment is not necessary in my experience but mandatory when doing the opposite.
To achieve a better yield of gel extraction, prefer low melting agarose to regular agarose as it dissolve better. Also, I would lower the the insert-to-vector ratio that seems to me quite high. Usually, 3:1 ratio works well.
Best
Nick
Hi patrick,
It is hard to tell concentrations of different size fragments in a gel as the longmer traps more EtBr so fluoresces better so quite a weak signal of shortmer ( or in other methods primer-dimer) can be quite a lot of material. Good luck,
Paul
Hi Patrick,
Both Paul and Nicolas had given good advice. I would also recommend going back one step to purify your fragment instead of wasting your time screening many colonies for your correct clone.
My only thought to add on this is do you know if the small fragment has both the ends? If it only has one of the ends then you will have a chance of getting your insert. The presence of the other fragment however will still interfere and could be cloned along with you fragment. So your success rate will be reduced.
-Richard
I would guess that among the transformants 250 bp insertw will outnumber the 2.5kb by about 5:1, but if you screen enough recombinants, you'll probably get your insert. Watch out for 2500 + 250 double inserts.
Thanks you for your suggestions. @Richard: Both fragments have each restriction site, unfortunately. @Andrew: Is 5:1 an empirical ratio, or is there a way to calculate expected insert ratios? Luckily, 2500+250 double inserts are not possible in my case because both fragments as well as the plasmid have all the same two restriction sites each.
5:1 is a guesstimate based on experience. It is probably possible to calculate the ratios theoretically, but the physical chemistry would be dauntingly complex.
Something just struck me.
Unless the fragment is gel purified there will still be the original plasmid backbone present in the ligation reaction. This will strongly affect any ligations.
-Richard
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