Hi All,
I've been working with a 150 bp fragment for PCR amplification of a repeatitive element LINE on bisulfite treated DNA to send off for pyrosequencing. I'm getting low yeild of amplicon so when I send it out to sequencing I get a low signal for my pyrogram. I've optimized my annealing temperature and magnesium concentration so now if I lower annealing and or increase the magnesium concentration I get non specific products. I have tried increasing the amount of template (6x more) added to the PCR reaction and yielded little to no increase in PCR product.
Finally I figured I would try to re-amplify my fragment using my PCR product from the first round. At this point I got a strange result, smeared bands at high molecular weight with no band at where my product was. I tried doing a clean up of the pcr before I did the re-PCR as well as tried cleaning up the re-PCR amplicon with a PCR clean up kit but the smears still stayed and it looks like theres a tight band stuck up in the wells.
Any suggestions?
Three options I can see:
1. not all (in my case the most) PCRs cannot be re-amplifide in a good way. Often you get smears like you mentioned. Often it doesn't help to clean up the amplicon from the first round.
2. the only chance I can see actually is to dilute the first round product 1: 10, 1:100 and 1:1000 and try again, perhaps 1:10.000 as well.
3. to create new primers which are a bit out of the sequence you wish (around 20 - 40 bp on each strand) and do the first round with that and the second round with your original primers.
Then you might be lucky to get a nice product in the right size without a smear.
Best wishes and good luck
Sven
Few Suggestion
1. Change the Taq.
2. Increase the reaction volume with high DNA input(4X-5X).
3. Bead purify (2X of bead) the 1st pcr product and repeat PCR with complete purified product by giving only 5-6 cycle of PCR.
4.You can also try nested PCR as suggested by Sven.
yes, unfortunately you have to invest in some new primer pair. I tried to reamplify with the same primers several times. It never worked. For whatever reason, you have to do nested pcr in such cases.
Oh no, in some cases it work very well but in some not at all using the same primer pair from the first round. This in my hands, I meant. My experience is that you have to dilute the first round cDNA not to overload the secound round PCR. Sometimes it works much better. I do it very often to get a better signal for seuqence analysis.
Interesting, with my pcrs, even if the primer worked perfectly, and even if I did dilution series, it never worked and I couldn't figure out, because theoretically, you have everything there for a pcr ... my boss told me the same, that you have to use nested. He told me at that time, it is sometimes enough to just do one side nested (design only one additional primer for the nested and use the other from the original primer pair).
Both might be possible but you can never be sure. It is always try and error conneted with a lot of additional preparative work, unfortunataly. At the end you also might not be successful which makes it very frustarting. :-)
Dear Dr. Morgan, First of all try the following proportions for your Master Mix preparation:
2X Mix 91%
F primer 4%
R primer 4%
Taq 1%
I think re-amplify PCR products is not a good idea. You must concentrate on getting good PCR product from the first time. Try to design your primers, measure your genomic DNA and try to repeat extraction from a new sample to get the suitable quality before doing PCR.
If the goal is to get enough of your PCR product to sequence I would suggest:
1) Increasing the reaction volume in each PCR tube. I've had great success with 50 microliter reactions
2) Send in the maximum volume possible for sequencing. Don't do a cleanup unless you have multiple bands or really bright primer dimers since you will loose product during a cleanup.
If neither of those strategies work, clone your PCR product into a plasmid vector and sequence from that.
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