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About PCR smear - Anyone can help me? (Feb/07/2006 )

Hi Everybody:

Good evening!

Recently, I use a pair of primers to amplify a gene fragment, I have succeeded to amplify this fragment, and I repeat it mquite a few times. Now I want to add XhoI Sites to 2 ends of this fragment. when I design the primers containing XhoI Sites on the 5'ends. And I employ the purified DNA fragment (PCR product ) as Template, I get nothing but smear. PCR RXn program is as same as before. 94C 3min, Then 35cycles of 94C 20sec, 60C 30sec, 72C, 4min, then 72C for entensive elongation.

To mu supprise, I also do a tube as a control, which has the same Rxn componets except does not contain Final Conc. 5% DMSO, then the PCR product is an unspecific bands which is much smaller than the bands of interest, there is no smear.

Anyone can help me?

any suggestion is appreciated!

Many Thanks.

Cinba

-Cinba-

what is your homology between primers? that band you see w/out DMSO could be primer-dimer

why not amplify from the original template, to get the fragment with XhoI sites on either end? all that would be required would be to make a version of your original primers with the XhoI sites incorporated into the sequence

-aimikins-

QUOTE (aimikins @ Feb 8 2006, 02:04 AM)
what is your homology between primers? that band you see w/out DMSO could be primer-dimer

why not amplify from the original template, to get the fragment with XhoI sites on either end? all that would be required would be to make a version of your original primers with the XhoI sites incorporated into the sequence


Hi Aimikins:

Thanks for your help.

The homology between the forward primer and reverse primer is very low except the XhoI sites.

I use the the genomic DNA as template to amplify the fragment again, I add final conc. 5%
DMSO to the RXNs system. but a long smear appears. by the way, I improve the annealing Temp by 2 C. what a cumbersome problem!

-Cinba-

hi
5%DMSO is very high...
I use 1% and it's ok. I've read in triple master FAQ that more than 2%DMSO drives non specificity...
You may try with 1 1.5 and 2%.

-fred_33-

HI Fred 33:

Thanks for your reply. I find out the problem. Its is because of my primer.

best regards

-Cinba-