Lots of colonies but they're all blue! - Do I need to adjust ligation ratios? (May/03/2007 )
I'm pulling my hair out trying to ligate this PCR product into a TA vector! It should be the simplest thing in the world but I'm having lots of bad luck!
The vector is pCR2.1 (or is it called pCRDNA2.1? It's by Invitrogen) a simple TA vector with blue/white selection on ampicillin. I assume the ends are dephosphorylated.
When I transform, I get plenty of colonies but they are nearly all blue. A colony screen (M13 PCR) shows a too-small product, probably empty vector, for all the white colonies and the blue ones that I also screen.
So: decent transformation efficiency, but the vectors are... self ligating?
My ligation reactions are typically:
2ul buffer (5x)
0.5ul T4 ligase
Ligate 5 mins @ room temp, transform 4ul
So, perhaps my insert:vector ratio is not high enough? The insert is a PCR product cleaned from a gel. Is it simply a matter of trying different ratios?
Edit: I should mention that this is the first time using pCRDNA 2.1 - I normally use pGEM-T but I ran out.... I don't know how old this tube of pCRDNA 2.1 is. So far I don't like this cloning vector very much - it's given me nothing but trouble
I haven't TA clone for several years, but if I'm right that is a vector with sticky ends and If that is true, then I have the answer to it. the problem is that the insert poly A's tail is loose during the gel cleaning, so there is no way that a ligation can occur. You don't need to clean the insert if there is only one PCR product.
Just to be sure, you are using Taq polymerase to make the PCR product right? No proof reading polymerase here.
Do you know how much insert you are adding? COuld there be problems with product recovery from the clean up kit?
If your PCR reaction clean (only a single band (your product), and free from any secondary bands), it might be an idea to directly TA clone your PCR product. The longer the PCR products waits, the more likely it will start loosing the A overhang.
Also, is the T4 ligase in particular and the ligase buffer still in good condition? The T4 ligase goes off fairly easily and the Ligase buffer doesn't like freeze thaw cycles. The ATP in the buffer goes off.
Since this is TA cloning, the vector has a T overhang, which prevents religation.
A list of stuff on TA cloning
TA cloning relies on the Taq polymerase adding extra A 3' to the PCR product.
Taq only adds on average only a single A to 70% of the PCR products that it makes.
Most people prefer using fresh PCR product (also recommended by protocol) as the A overhang falls off after a while.
Proof reading polymerase product blunt end PCR products.
You might also test some of the blue colonies for an insert. Depending on what you are cloning, expression of active LacZa can occur even with an insertion.
That so true. If the insert is small and by some bit of luck in frame with the lacZ gene. After all the N terminus of the LacZ protein isn't very important, thus allowing the insertion of the multiple cloning site into the gene in the first place.
I wish I didn't have to gel-purify! Unfortunately I've not been having much luck with my PCR the last few weeks and I often get multiple bands (if you recognise my user name, I'm still working on that darn Assembly PCR - it worked so well the first few times!)
I always screen all my white colonies and a few blue ones for good measure. When I was using proofreading enzyme for my assembly PCR and ligating into pCRBLUNT I had to screen lots of blue ones because my insert is exactly 88 codons (so LacZ is still in-frame). With a TA vector it's not in-frame, but I still check.
The ligase is pretty recent and I tiny aliquots of ligase buffer (in PCR tubes) to minimize repeated freezing. In any case, it appears that the ligation is working to close the TA vector...
It seems that my PCR product probably just doesn't have any A tails. It should though;
There's a paper where they characterised that (you've probably seen it). I got all excited thinking I'd found my problem, but no, I had a brief look and my primers are fairly close to the consensus published in that paper, so it really should have A tails.
Maybe it lost the A tails? Perhaps, but I've tried repeated PCR attempts and it doesn't seem to fix things.
Anyway it seems like I've thought of all the same things as you guys... thinking about it again now it's probably something to do with my really poor PCR results rather than ligation conditions. It's a bad sign when my samples get a band and there's nothing in the negative but nothing in the positive control either!
Well, thanks for your input!
When using commercial kit, usually you will have good chance of ligating. Check through invitrogen and let them know.
Though it shouldnt self ligate. But somehow it is really hard to judge. If the problem still persist, perhaps you can check your PCR products. Sequence it or something like that. Yea.... I agree with you. I am suspecting your PCR products instead. Cheers.
if the vector is old or has been badly stored, it may have lost many of its T overhangs. Otherwise, since you say you must gel purify your PCR product, do you have enough DNA left after the purification? Or, try adding the A overhangs after the cleanup?
I don't know if I'll have time to revisit that ligation (thesis due in 2 weeks), but other people have been having trouble with PCR so that's probably the cause...
Next time I do cloning, I'm going to do sticky-end PCR!! No digestions and no TA vectors!
Haha.. all the best for your thesis. Erm... i have to say TA cloning is easy to do compared to others.