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Primer Decontamination - (Sep/20/2010 )

Hi,
Is there anyway to get rid of primer contamination? I have a band in negative control that correspondes to the size of the required band. Changed every thing except the primers, but still the band is there which makes sense that the primers are contaminated. Please help. Thanks

-Bilal Malik-

Bilal Malik on Mon Sep 20 12:53:44 2010 said:


Hi,
Is there anyway to get rid of primer contamination? I have a band in negative control that correspondes to the size of the required band. Changed every thing except the primers, but still the band is there which makes sense that the primers are contaminated. Please help. Thanks


Before we go to that..

Might this be a primer dimer?

Could the template be contaminated? Are you getting a signal even when a template is absent.

Could the insides of your pipette barrel be contaminated? Do you use filter tips?

If the primers are contaminated, there is nothing you can do. Bin the primers and buy new ones.

Next time, prepare your primers as two stocks. A master stock at 100uM and a working stock at 10uM
The primer master stock is guarded with absolute care. You use only brand new TE, new filter tip to prepare this stock.

The working stock is made by diluting a sample (10ul) of the main stock with distilled water. This working stock is the tube which you actually work with. If this tube gets contaminated, you can easily discard it.

Contamination happens, will happen. That is why you must aliquot all your PCR reagents from dNTP, to PCR buffer, to MgCl2 solution so that you can throw everything away the moment contamination is believed to have occurred.

-perneseblue-

Thanks a lot for replying. Well no itís not the primer dimer as the band I am getting in the -ve control is similar to the band required (402bp). Secondly I am getting the band even when I have just worked with the -ve control only (no template added) . As far as tips are concerned. Yes I am using the filtered tips so there is no question of pipette based contamination. On the other hand I always make aliquots of the reagents but in this case this contamination thing has happened the 1st time I have set up the PCR (did not even gave me a chance to aliquot the reagents). I though there would be kit (spin column) to separate the larger bands from the smaller ones -_-.

-Bilal Malik-