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[ocean_sky83]

When I run my gel, I noticed a very faint band, I almost missed it. I do not know yet  whether it could be because of "carry-over" from the well beside it. If it is not due to accidental carry over, I want to do some optimising to my pcr, my question is,

a) How do u tell if your bacteria dna has high GC content? How much of betaine do u usually add to your reaction tube?
If I do use betaine, is it necessary to lower or increase the annealing temperature?

I hope I am thinking too much about the faint band. It is so faint that I stared very long at the image before realizing it Do you usually follow up on an extremely faint band or just rule it out as negative, assuming you do not know whether in first place it is supposed to be present. When I observed the other positive samples I have, they are all very bright clear band. There was one faint like a "pencil line band". Could this be due to high GC content? My product size is about 700+bp I run it on a 1.5% gel.

Usually, would you repeat on such cases?

Any advices very much appreciated!!
Have a great day!

Thanks!!
Mich

Edited by ocean_sky83, 08 October 2010 - 08:22 AM.

[Adrian K]

Hi Mich,
Not sure if this helps.
I deal with high GC dna content bacteria, and I add 5% DMSO into it, never use betaine before.
If the faint band is something I really wanted, I will do follow up for sure by optimizing my PCR. Usually I will start with temperature gradient before I try other parameters.

I got no idea how exactly you mean by extremely faint. There used to be a case for myself where the amount is too little, I pool a few tubes, gel excise the band and clone it into cloning vector and sequence it. Of course, you must ask yourself what are you going to do with this band.

Adrian