5 replies to this topic

[Moebius]

Hi. I was wondering why sometimes I seem to get PCR product using the same protocol and other times nothing shows up. The DNA that I use is a linear plasmid DNA cut with MssI and has been in storage for 2-3 years. I was wondering if there is any other factor other than degradation? I vortex and spin down the DNA. I am not sure what is wrong. Thanks.

Any help would be appreciated.

[SOS response]

Hey, vortexing is not a good idea, because it breaks DNA, centrifugation shouldn't be a problem...

Edited by SOS response, 14 March 2011 - 01:45 PM.

[Moebius]

SOS response, on 14 March 2011 - 01:44 PM, said:

Hey, vortexing is not a good idea, because it breaks DNA, centrifugation shouldn't be a problem...

Thank you. I was wondering if Plasmid DNA could also be affected. The reason I asked was the DNA I am using is 2-3 years old. Should I be extra careful and just flick it before adding to my PCR tube? Thank you.

[phage434]

A common problem is using too much DNA in your PCR reaction.  Try reducing the amounts used 10x or 100x.

[leelee]

I doubt the vortexing is a problem, I vortex my DNA all the time and I've never had an issue.

[Moebius]

phage434, on 14 March 2011 - 06:26 PM, said:

A common problem is using too much DNA in your PCR reaction.  Try reducing the amounts used 10x or 100x.

Hi Phage,

I was wondering how much is "too much" DNA and what are the effects of having too much? Thnaks!