pcr

[315]

hello I have nonspecific band. my gen is 637bp but i have 700bp band.

[SebaQ]

First of all, are you using high resolution agarose, polyacrylamide or you sequenced you pcr product? Because in normal agarose sometimes it's hard to see a difference between 637 and 700 bp.

Anyway, you could give us some clues of what you're doing, 'cause there are many reasons why you could have an inespecific band.

1.- If you are using a high concentration of template DNA, you can have an inespecific product.

2.- If your primer concentration is too high, you can sometimes "force" them to anneal to something unwanted

3.- If your primer GC content is too low, your primers can anneal to undesired sequences.

4.- If your annealing temperature is not the optimum, primers can anneal to inespecific sequences.

And so on... check this things first, 'cause they are the most common reasons to have inespecific bands.

[315]

hello I have nonspecific band.
tm F primer: 59.87 , GC content:32.0%
tm R primer:60.55 , GC content:41.4%

concentration primer:0.5Ml(macrol liter)(20pmoles/Ml)
Annelling tm:48
my protocol:
Taq polymerase:1U
dNTP:250Mm(macro mole)
MgCl2:1.5mM(mili molar)
buffer 10X:0.1X
Template:1macro
total volume:25

my product :1187bp

[SebaQ]

Still some things missing,

First you said you needed a 637 bp PCR product, and now it's 1187 bp lenght.

Anyway by looking at that, you do have a very low annealing temperature. If your primers have Tm of nearly 60ºC, you can totally rise the annealing temperature up to 62-63ºC without disturbing the reaction so much.

Try a gradient PCR to attack the problem

[Alicialsw]

I believe you should increase the annealing temperature as SebaQ mentioned. You can also try to add some additives like DMSO

Hope it helps

[315]

Hi, 1187bp is another gen. before I got multiband in 48 anneling temperature but now I had no product even at 50-60 annelling temperature.

[SebaQ]

If that's true and there's no other problem associated, you better excise the band you want to work with.