PCR problems on a ligation product - (May/18/2009 )

Hi! I have a ligation product composed of three pieces and which is about 4kb in size. I only get a small amount of this ligated product and so the next approach is to amplify the ligated product using PCR. The problem is, I can't seem to amplify the linear DNA fragment using the outside flanking primers. I have tried a nested PCR, in which I tried to amplify each of the piece and I got PCR products, of about the right size for each of my pieces. What seems to be the problem here? Thank you very much for the help.

ironman on May 19 2009, 10:53 AM said:

We need your reaction conditions before we can help...

swanny on May 18 2009, 05:59 PM said:

Thanks. The PCR reaction consists of:

5 microL buffer
4 microL dNTPs
1 microL primer1
1 microL primer 2
37.75 microL H20
0.25 microL ExTaq
1 microL template (my ligation product)

The PCR running conditions are:

94 degC- 2mins
94 degC- 30 secs
50 degC- 45 secs (annealing temp and time- i have rechecked this)
72 degC- 4 mins (extension time; polymerase activity- 1 kb/min)
4 degC

Ran for 30 cycles...

Thank you.

ironman on May 18 2009, 06:18 PM said:

I'd just like to add that I have also tried clone the ligated fragments into a vector, but I was not successful. The thing is, when I try to amplify each piece in the ligated product (just to check their presence), I am getting PCR products, a possible indication that they are present in that DNA fragment. Is it possible that they have not really ligated. The ligation reaction was gel-purified. I extracted the band which corresponded to the approximate size of my expected ligated product.

ironman on May 19 2009, 12:42 PM said:

OK, a few questions:
What length of overhang do you have between each of the three pieces before you try to ligate them to each other??
How long, what temp do you use of your ligase reaction?
How do you prepare the fragments for the ligation, after PCR amplification?
Do you know if your ligase is working? Test it on some DNA ladder; if you don't get the ladder shifting up with a 20 minute, RT reaction, the ligation step is failing.

What length of overhang do you have between each of the three pieces before you try to ligate them to each other??
How long, what temp do you use of your ligase reaction?
How do you prepare the fragments for the ligation, after PCR amplification?
Do you know if your ligase is working? Test it on some DNA ladder; if you don't get the ladder shifting up with a 20 minute, RT reaction, the ligation step is failing.

I have about 5bp overhang for the three pieces.
Usually I allow the ligation reaction to run overnight at 4 degC. If I run it for 2 hours, the reaction is at 20-25 degC.
After PCR amp, I run the mixture in agarose gel electrohoresis. I use Qiagen Gel Extraction kit to extract DNA from the gel band with the expected size.
I believe the ligase and buffer are working because we have used it in other reactions with no problem at all. And a band corresponding to the right size of the three pieces ligated together is showing up. I know this is not conclusive, that's why I tried to amplify each of the piece using the ligated product as the template and I got all the three pieces amplified.

Thanks.