Multiple ligations - (Sep/26/2006 )
I know I posted a while ago, but I am still struggling with a multiple fragment ligation.
I have three fragments that I have obtained through PCR that need to be ligated together (1+2+3). They have appropriate restrictions sites to do this, and I believe my digestions are working properly, because I do see some ligation product.
Once I get the three ligated, I planned on using it as a template for a PCR (using the forward primer from fragment #1, and the reverse primer from fragment #3). I obtained ligation product, gel extracted the right band (because there were some 1+2 and 2+3 bands), but consistently am not seeing PCR product. I tried gradient PCR to try a variety of melting temperatures as well.
My question is, in your experiences, how do you usually approach such a situation? Sequential ligation? Do you purify after ligation before PCR, or simply heat inactivate the ligase, or use it as is? I am concerned that gel extraction might be having some effect on the fragment, or it is not "clean" enough to use as a PCR template.
I'm open to suggestions -- what do you guys usually do to ligate multiple fragments?
As I mentioned in the earlier thread, I throw my multiway into a vector. Do a large scale screen to find a clone with the right ligation product. Midiprep that clone and proceed from there. This manner I make a lot of DNA to play with.
As for why the PCR, I have 2 thoughts
1- The DNA is contaminated with salts and buffer from the extraction process. If that is correct ethanol percipitation should correct the problem.
2- Could there be a problem with the polymerase. the DNA fragment is simply too long or hard for the polymerase to amplify? In which case a change in PCR conditions is needed.
OK, we're all guessing, but I'd guess that you are using way too much of your ligated DNA in the pcr reaction. All you really need to do is to put a pipet tip into the gel at the right spot and then contaminate your pcr reaction with that tip. Any more, and you are just making problems for yourself. Or, avoid the gel entirely, dilute 100x or more and add 0.3 ul to your pcr reaction (smallest amount you can pipet and still verify that there is some).
You could try this two at a time, since you already have the primers.
Have you thought of fusing them by PCR? You can design primers such that they overlap...so reverse primer from fragment 1, overlaps with forward primer from fragment 2. Reverse primer from fragment 2 overlaps with forward primer from fragment 3. You mix the 3 together and do 5 cycles of PCR, then add the forward primers from fragment 1 and reverse primer from fragment 3 and amplify for 25 cycles. The overlapping bits are added on, so you add the reverse complement of forward from fragment 2 to the reverse primer of fragment 1 and so on.