LR PCR and running gels - Roche Expand Long Range dNTPack (Aug/09/2008 )
Hello..
I've been using Roche's Expand Long Range dNTPack to amplify between 15 and 20kb. I'm still working on optimization but was wondering if anyone has had success using the DMSO provided? Or any other tips? I'm getting decent results after optimizing the annealing temp but trying to make it more robust.
Also, a question about running these 15kb and 20kb products on gels. I've tried running the products on 0.7% agarose gels. I find that most of the product gets stuck in the wells and won't run down. Does anyone have any suggestions? I've tried heating the sample to 95C for 5 mins to denature it then putting it immediately on ice before loading it...and have lowered the amount that I load but still no good results. I've been running them at 120V for 60 mins and the products still stay in the wells while the ladder runs down normally.
Any other ideas would be appreciated.
Thanks...
Have you been loading your samples with a reducing buffer?
I've never heard of DNA not being pulled from the well. If it's DNA it's loaded with negative charges. Perhaps your samples don't actually contain any DNA??
Hi,
I am also starting to work with long range PCR and the truth is that is horrible. I also use the kit from Roche. Until now I could not to amplify with a lot of efficiency, I always get pcr products very tenuous. Sometimes I also had large fragments on the wells, the solution is to use a lower voltage (50 V).
Good Luck
I am also starting to work with long range PCR and the truth is that is horrible. I also use the kit from Roche. Until now I could not to amplify with a lot of efficiency, I always get pcr products very tenuous. Sometimes I also had large fragments on the wells, the solution is to use a lower voltage (50 V).
Good Luck
Thanks for the replies. I have tried lowering the voltage to 80 (and will now try 50) and have also tried diluting the samples and heating them to denature them. I am positive that there is dna...the wells are very bright so all of the dna is getting stuck in there. Some of the dna goes down on a few of the samples and since I'm using this 1kb extension ladder that has bands at 15 and 20kb, I know that it is the right size.
However, most are still stuck in the wells. I'll try lowering the voltage. Has anyone else had this problem? Could it be a problem with the agarose itself? I'm using Seakem.
Thanks...
I've never heard of DNA not being pulled from the well. If it's DNA it's loaded with negative charges. Perhaps your samples don't actually contain any DNA??
I haven't been using reducing buffer...but, honestly, I've never heard of anyone using it. I looked it up and read that reducing agents are used to "reduce" the secondary and tertiary structures of proteins by breaking disulfide bonds. Since I'm not working with proteins I hadn't thought of using that. However, since I assume that such long pcr products can form 'complexes,' I did try heating to 95 C to denature it...to no avail.
Also, the reducing buffers that I've read about contain SDS which gives a positive charge to all proteins...which I don't want.
Some hints for electrophoresis:
-use high quality agarose (NOT from a cheap supplier! yes there are differences!)
-use low percentage of gel (I use 0.5%)
-Use very low voltage (50V especially at the beginning, I always start with 30V for 15min than go up to 50V)
-do not overload your gel (use little sample)
