Marker

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Feb 24 05:31:19 EST 2000


In article <Pine.OSF.3.96.1000224115918.5740B-100000 at USTCC.ust.edu.ph>,
Ma Sheila M. De Jesus <mshmdeje at USTCC.ust.edu.ph> writes
>e are a little new to this endeavor, howver, when I run PCR on ATCC E.coli, the 
>gel showed 2 bands: 1st band between 1330-1584 and the second band near 983. The 
>Dna isolation i used is the genereleaser protocol.  Since I used the universal 
>primers on this, I expect only the1st band.  Could the second band be a 
>contaminant?

Unlikely to be a contaminant, more likely to be false priming either
within the within the 16srRNA gene or the primers are priming elsewhere
on the E.coli genome.

To test. Take the 1330-1584 product and re-PCR 1ul of a 1/1000diln with
the same primers for say 20 cycles. If the smaller band re-appears then
you know it is false priming from the rRNA. If not it is from the
original xsomal.

You can then try increasing the Tm such that you only get your wanted
band. What about doing a blast search on the E.coli genome with your
primers?

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk




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