5' race - (Apr/28/2011 )
Hi all
i have been doing 5'race for the last 1 month and i am really frustrated as I get my band but with lots of smear in background. I used nested primer also but still the smear saga continues. I using pcr product as a template. I diluted it to 10^4 times then also i got smear(faint)but faint amplification of the desired band was also there. I tried using pcr additives but nothing works.I wonder why that smear continues even though i use so diluted template, further with the same dilution there is smear only when i use nested primer designed just a few bp to the 3' end of the first primer.
PLease help me out as I am abt to become mad.
Thanks in advance
It's been a while, but when I did RACE I gel purified the bands then re-amplified them to get clean bands. I think getting smears is not uncommon for RACE.
deespike on Thu Apr 28 14:55:42 2011 said:
Hi all
i have been doing 5'race for the last 1 month and i am really frustrated as I get my band but with lots of smear in background. I used nested primer also but still the smear saga continues. I using pcr product as a template. I diluted it to 10^4 times then also i got smear(faint)but faint amplification of the desired band was also there. I tried using pcr additives but nothing works.I wonder why that smear continues even though i use so diluted template, further with the same dilution there is smear only when i use nested primer designed just a few bp to the 3' end of the first primer.
PLease help me out as I am abt to become mad.
Thanks in advance
As said before smear is a common problem in RACE. I have spent months for one band. However i can recommend you something. probably you have tried varying MgCl levels, varying DNA dilutions, DMSO and varying annealing temperatures etc. If these didnot solve the smear, try something on your PCR cycles. Sometimes some primer couples give smear in longer elongations. For example do not add 20 seconds for each elongation cycle (which is recommended in RACE protocol). Perform an ordinary nested PCR. Just predict your length and elongation time and fix the duration, do not extend it.
Also purifying at first step may help. If you gain an uncertain band in first PCR, you can gel purify it and take into second PCR. But if you are not sure which band are you looking for, do not select this band, because yours may be elsewhere. In one of my cases I gained three distinct bands after 2 PCR and these were not smaller than the band I have gained in first PCR. So I cloned all of them and sequenced, then I could find wihch was of my ineterest.
Also one of the last solutions can be ordering different primers, which are far enough, hard to form dimers and also long enough for specifity.