5 replies to this topic

[jamestoon1]

I tried to amplify a region, but here's what I got:

The band size of interest is 200bp and I got a lot of smear (should i call it smear because it has specific discrete pattern).


Here the details of the PCR:

2X PCR master mix ---> 10 ul
Forward primer (10 pmol) ---> 0.5 ul
Reverse primer (10 pmol) ---> 0.5 ul
Template DNA ---> 0.5 ul

I have tried diluting the template DNA, but I still get all those smears (i.e. when I diluted the template, both the band of interest and the smear become faint)

I don't think the problem lies on the primers, because
(1) there has been many previous publications utilizing this primer pair
(2) i thought probably my primers degraded or something like that, so I tried to order new sets of primer (same sequence, but newly synthesized one) from different companies

-----------------------------------------------------------------------

Initial denaturation (95°C, 2 minutes)

35 cycles:
Denaturation (94°C, 30 seconds)
Primer annealing (60°C for 45 seconds)
Elongation (72°C, 1 minute)

Final extension (72°C, 5 minutes)

-----------------------------------------------------------------------

Can anyone help? Thanks in advance~

[hobglobin]

What type of ladder is it? If the upper band in every lane is 200 bp and the smear 100 bps and below, then this smear might be primer dimers and also amplification products of these dimers, not nice but you'd have your band of interest at least...but that's a guess, because no idea about the real band sizes.

[jamestoon1]

Hi. Thanks for the reply. It was 100 bp ladder.
The band of interest was there, but the primer dimers are soooooo much more pronounced than the band of interest.
I have also tried (1) touchdown pcr, (2) increasing the annealing temp, (3) adjusting (increasing/decreasing) the duration of denaturation, annealing, extension, but all gave me similar results.
Can anyone advise?

[Ameya P]

Hi James,

Did you run a No template control along with this PCR? I am just guessing if these smears are directly from your template DNA. What is the source of DNA?

[hobglobin]

To check the primers you can reduce the primer concentration too e.g. down to 0.1 uM (you have now a concentration of 0.25 uM if you have 20 ul total volume per tube). If the smear becomes less it's perhaps a primer-dimer problem (they can also be amplified, if they overlap in a way that the polymerase can attach; this can happen with badly designed primers).
And try out AmeyaP's idea of course.

[jamestoon1]

Ameya P, on 23 November 2012 - 10:57 PM, said:

Hi James,

Did you run a No template control along with this PCR? I am just guessing if these smears are directly from your template DNA. What is the source of DNA?

hobglobin, on 24 November 2012 - 09:12 AM, said:

To check the primers you can reduce the primer concentration too e.g. down to 0.1 uM (you have now a concentration of 0.25 uM if you have 20 ul total volume per tube). If the smear becomes less it's perhaps a primer-dimer problem (they can also be amplified, if they overlap in a way that the polymerase can attach; this can happen with badly designed primers).
And try out AmeyaP's idea of course.

Thanks for the suggestions. Will try them and let u know the result.