colony PCR - (Aug/03/2009 )

Hi all,
My colony PCR gel shows required amplicon along with some high mol.wt bands. what could those be.
thanX

novagen on Aug 3 2009, 10:17 AM said:

Possibly bacterial genomic DNA that you used as the template.

Hi
you mean to say bacterial genomic DNA has some homology to my Primers.
Thanx

novagen on Aug 4 2009, 11:40 AM said:

If you run your PCR under loose condition (low annealing temperature), there are chances for your primers to bind to genomic DNA region, even close (to some extent) in sequence to your target. As my experience, sometimes they even overweighted my target T_T (in this case, my primer design was not good)

It's probably just your vector and or genomic DNA - not necessarily amplified, just present on the gel!! You see this with mini-prepped samples too, usually 2 bands (supercoiled and non-coiled plasmid) and sometimes a blur of gDNA at the top!!

Hi there

I have the same problem too! When i perform colony pcr, i get many bands......of higher molecular wt than my expected band. Do u think it is contamination due to too many cycles? or could it be the condition of pcr itself?

Ms new scientist on Aug 25 2009, 06:55 AM said:

It's the normal situation. You can increase the annealing temperature more 3-5 degree to obtain more specific products. Also, 25 cycles is enough. Hope this helps ^^

Sample preparation can also help clean up the gel. I pick my cells into ~150 ul TE and disperse the cells, then I use 1ul in the PCR reaction. I have found much cleaner amplification and better consistency.

Ms new scientist on Aug 25 2009, 05:55 AM said:

I used to have same experience with you. After I diluted my colony template (10x), the extreme high mw band disappeared.