Whole mtDNA genome amplification with long-range PCR...trouble - (Jan/16/2014 )
Wondering if any of you have amplified whole mtDNA using long-range PCR? I'm having a small problem and would love to hear from someone who has done this before. So the organism I'm working with has a mtDNA genome of about 15 kbp. I designed primers which are close together (500 bp) with hopes they would do nearly the whole genome in one pcr (which has been done in my lab on other taxa using other primers). I wanted to be able to test the primers to make sure the priming sites were good before attempting the long range, so I also ordered the reverse complements of both so that I could amplify the 500 bp fragment between them, using standard pcr, as a test of their priming ability. So, the test of my primers worked wonderfully, so I'm sure the priming sites are good, but when I switch to long-range PCR I just get a smear. The DNA should be good, it was freshly extracted using a Qiagen kit and the samples were not stored (EtOH) long before extraction. So my question is, anybody have any protocol tweaking experience that worked? I'll add one thing, the smear doesn't have even a hint of a band and doesn't appear to be the kind that raising the annealing temperature would help. Thanks for any input you can provide.
P.S. Image is of 2 samples and a slightly overloaded hindi III ladder.
In long range PCR you cannot avoid non specific amplification. I have got similar results. You can try to elute the amplicon in E-gel or low EEO. You can also try ExoSap treatment or PCR purification Kit.
IndianScientist, thanks for the reply. Since there is no visible band I'm not sure that a ExoSAP treatment or PCR purification would help. It would definitely get rid of some of the smear but since there is not visible target product I don't think that would get me any further ahead. I'm wondering if possibly tweaking the PCR conditions could help produce my target band (since I know that both the DNA and primers are good). I realize that getting extremely large amplicons is going to be troublesome period, but hopefully someone out there has found a workaround for this problem. Cheers!
whatever you are seeing is an amplicon even though it has not came out of the well. One possible rreason maybe the poresize.. try to run it in 0.8%. In which transilluminator you are looking it..??
If it is a BIORAD you can quantify it. Also try and load the template adjacent to your PCR product. So that you will have a better idea. If you want to isolate mitochondrial DNA then i have protocols for it. Do let me know if u want it..
Could you please tell us the details of your PCR?
template, reaction mix, enzyme
Thanks for the reply guys. The PCR and reaction conditions are as follows on image:
That image does not download for me.
I cannot see your reaction conditions either. But looking your image, my first response would be to increase annealing temperature in steps of 2-3 oC till you start bands of some sort. 1 kb, 2 kb, anything at this point in time. Once you reach there, then, I would play around with annealing times and see if I can get my hands on some type of PCR enhancers to get a the desired product.