i am trying to identifies unknown sequence in nitrate reductase gene from chlorella vulgaris using anchored pcr.
can someone help me with some specific protocol?
thanks a lot.
I do not know this technique but given that the first extension is single primer extension it has no fixed stopping point so must generate a huge number of different size extension products. These will all be ligated to the reverse primer so you must always get a smear on second round amplification. This smear product should be cloned and clones sequenced to give different length products containing your gene (partial) sequences
Hi Najib,
why don't you try at final PCR to increase a little bit the Tm (I don't know, 2 or 3 degrees) to reduce false priming and amplification and be more specific?
the number of cycles is also critical, more you'll have cycles and more background you'll have. with a prior PCR, I would use 1/100 as you do but with 30 cycles.
fred
Hi Fred,
I do not understand how this helps. If the first round is limited by only when the polymerase falls off then we will have a range of sizes which get ligated to a reverse primer. PCR between forward ( specific) and ligated reverse primer should still produce a smear of sizes after a second round pcr
Hi Paul,
the idea is to follow the protocol as it is described in this one (attached picture, even if it's not really the same goal).
I hope this helps
fred
Thank you for the very clear explanation Fred but I still have a problem in that in RACE the dna target is anchored at one end by polyA and at the other end by GSP so there should only be one (maybe a couple if alternatively spliced) size of bands which can be amplified. Najib has specified that the first amplification was with genomic dna and GSP so extension will happen until the enzyme falls off the template and then the reverse primer is not internal to the sequence but is added on at the ends so is not a fixed distance from the gsp but I will think more about this.thank you
Paul
Dear Paul and Fred, thanks for your answer.
i followed the protocol in these papers:
Article LT-RADE: An Efficient User-Friendly Genome Walking Method Ap...
Article LT-RADE: An Efficient User-Friendly Genome Walking Method Ap...
and after the tailing with TdT, i did pcr using GSP2 and AAP and using the pcr product i did the second pcr however i got smear as shown in the pic.
Hi Najib,
2 seems to be worst than 1....and it's very high.
you should taka back and look at the basis. what is the species you're working on and do you have the possibility to blast your primers?
blasting should give you an idea on your target and the specificity you need.
try primer blast at NCBI with the right organism, it should help you (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome).
fred
Hi Fred, i am working on chlorella vulgaris, a microalage, and i wanted to determine the promoter region of nitrate reductase gene. based on blast, i designed the primer. the basic of the anchored PCR is as in the figure:
Hi Najib
sure the nested PCR should have give you a clean band. I don't understand. you have the sequence, but did you try to blast GSP2 and GSP3 just to be sure you're sufficiently specific and how long are those primers?
fred
Hi Fred, for the GSp2 and GSP3 their size is around 20 mer . also i blasted them against the genome of chlorella vulgaris. furthermore, for nested pcr, i tried to used annealing temperature of 55 and 60 C.
najib
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