troubleshoot pcr problem

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ggkk
ggkk's picture
troubleshoot pcr problem

hii,
my gene of intrest is of 2.3kb which is to be amplified with a A, B primers .but there is very faint band coming just above 2.5kb. the same template , i used to amplify 400bp segment with primers A, C primers. the result was good.
do i need to use faint band pcr product as template for pcr.
but i have come to know that sometimes the second pcr product would not be our desired segment.
is it true.
plz find me a solution.

i need clone this 2.3 band.

thanks in advance.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi ggkk,
I am not sure if I understand your problem, so here is my stab at it. You need to purify a 2.3 kb fragment but you are getting a very faint 2.5 kb fragment in addition to the 2.3 kb fragment. If that is the case, then all you need to do is run you PCR on a gel long enough to separate the 2.3 kb from the 2.5 kb fragment, cut out the 2.3 kb fragment as best you can, and then clone it. You will need to screen a few colonies and ideally sequence a few to determine that you indeed cloned the 2.3 kb fragment and not the 2.5 kb. Since the 2.5 kb fragment is very faint most colonies should be the 2.3 kb fragment and not the 2.5 kb fragment. 
Hope this helps

Jason King
Jason King's picture
If you only have one band and

If you only have one band and it is too faint then I'd reduce the annealing temperature. How many cycles are you doing? How long are you extending for?

ggkk
ggkk's picture
hello,

hello,
i had only one faint band at 2.5kb instead at 2.3 band.
annealing temperature 40degrees, 2 min (i tried 1 min , but there is no faint band even)
elongation temp 72 degree ,2 min
 30 cycles. i tried even with repeating pcr with 1ul of  faint pcr product as template. there is no result.
 

Ivan Delgado
Ivan Delgado's picture
 

 
Hi ggkk,
Annealing at 40oC is too low. When you get amplification at such low temperatures it is typically due to non-specific amplification. I suggest you raise your annealing temperature to at least 52oC.
Likely your problem is with the elongation time. Typical Taq needs 60 seconds of elongation per 1.0 kb, so if you are using only 2 minutes you can only amplify 2 kb. I suggest you bring your elongation incubation to 3 minutes and try again.
 

Jason King
Jason King's picture
Yes, 40 degrees is very low.

Yes, 40 degrees is very low. I'm suprised you don't have a smear of PCR products at this temperature. As Ivan points out, 2 minutues is too short for 2.5Kb PCR products. I guess the reason you get a 2.5Kb band is that you have a very long annealing phase (2 mins) and some of the extension is going on at 40 degrees.
 
If you can, I'd order primers that allow you to use Tm of about 55 degrees (1 minute is fine) then extend for 3 minutes at 72 and do about 35 cycles. Further fine tuning could include Mg2+ concentration and template amount.

ggkk
ggkk's picture
hi,

hi,
primer a has tm of 47 degrees, primer b  tm is 52.2degrees . what should be aneealing temperature?

Jason King
Jason King's picture
I think I'd order a new

I think I'd order a new primer a (at least) but if you want to use these then I would start doing PCRs with a Tm of 2 degrees under the Tm of the lowest primer. ie. 45 degrees.

ggkk
ggkk's picture
thank you .

thank you .
i got good band at desired size.
one doubt.
i used gradient pcr at 42,43,44,45,46,47 annealing temperature. i think increasing annealing temperature increases specificity alone. but when i checked  on gel, the band intensity is increasing with temperature. nearly at 46 degress ,i found a band with good and more intensity. how could it possible.