Digest does not confirm presence of fragment - (Nov/10/2012 )

Hey guys,
I am new to this forum, however, I read countless of threads and judging from the posts I am pretty much convinced, you can help me

Here's the thing: I am trying to insert a PCR-amplified 0.5 kb fragment into a 4.9 kb vector. Both are cut with the same REs (NotI-HF and SalI-HF, NEB). A sequential digest of the vector confirmed that both enzymes work. The cutting sites for the fragment lie within the primer-sites. After the digest, I load every sample on a gel, let it run and do a gel purification afterwards, eluting the purified DNA in small amounts of water. Then, I start ligating with T4 ligase at 4 °C over night. Here, I tried different vector:fragment ratios, like 1:3, 1:5, 1:10, and 1:15. I also tried to ligate at room temperature for one to three hours and overall, it seemed that 1:3 at 4 °C over night worked best for me. Then, I do the heating transformation in XL-blue bacteria, plate them (10 % and 90 %) on LB-Amp, and usually see only few colonies the next day - let's say on the 90 % plate around 20 to 30, which is quite low, however somehow it seems to work.

After DNA isolation, I start the double digest and surprisingly, I only see the cut plasmid - no fragment. However, the plasmid runs at ~ 5.5 kb and not 4.9 kb which might imply that the fragment is present. When I do a PCR with these samples and the corresponding primers, I can clearly see a band at 0.5 kb (plasmid as a negative control shows no band).

So it seems the fragment is present but the digest does not work properly. My next idea was to look for a RE that only cuts within the fragment but not the vector and yeah, yesterday I found one so I would try this on monday but nevertheless, I would like to know what's wrong with the digest.

Anyone an idea?

One of RE is not working/have Mutation in it. Can try two separate RE reaction for plasmid with insert to detect which one.

Exactly my idea. I digested each DNA sample with both REs separately and found out that both enzymes work fine, showing a band around 5.5 kb for every sample. Maybe, a sequential digest (although shouldn't be required) might help?

I'd suggest that your PCR product is not being cut with the SalI enzyme. This is a known problem with this enzyme. It fails to cut PCR products efficiently. How many bases 5' of your cut site are present on your primer. Can you use another enzyme?

I checked this and indeed, there's literally no base upstream the recognition site for SalI. Now, that you mention it. i can remember, reading somewhere that SalI needs something like 15-20 bases for corrrect cutting. However, the vector offers also a SacII and BgII site so either, I start designing new primers or - maybe the faster way - I ligate the purified PCR product into pGemT, cut it with NotI and SalI (the additionanl NotI-Site in pGemT shouldn't display any problems, right?) and then ligate in my original vector.

One more thing: If SalI didn't cut the fragment correctly, how is it possible that the fragment seems to appear in the vector later on (I "confirmed" this with a PCR on the miniprepped DNA)? The only possibility may be that parts of the vector only contain the NotI-digest but not the SalI-digest. Since this seems to be a rather rare event, that would explain the small number of colonies on the plates.

I'd avoid SalI entirely if possible. I think the easiest and safest thing to do is to redesign your primers. You might want to avoid enzymes that cannot be heat killed. Both BamHI and BglII have that problem. NotI does not typically cut completely. SacI probably would be a good choice.