cloning faint bands - (Aug/20/2007 )

I am trying to clone a faint pcr band. My pcr amplifys a full length gene of interest plus a faint band that is a splice variant missing one exon. I need to clone the shorter faint band which is only 100 bp shorter than the full length band at 2kb. I am having difficulties because the band is so faint i lose it in gel extraction. I need the full length shorter version as i need to get it in an expression vector so can't redesign my primrs to only pick up the shorter version. any ideas??

Hi,

I guess you don't loose all of your shorter fragment, so you might try to reamplify it in a second round of PCR after having it isolated from the bigger fragment.
Don't know which enzyme you are using but you could use a proofreading enzym because two rounds of amplification might result in more errors in your PCR.

Cheers

A second amplification with nested primers or semi-nested primers would probably work. You can just try this by poking a pipet tip into the gel at the correct spot and transferring it to the PCR tube. No need to purify the band.

Another option would be to clone the larger band and then remove the exon you don't want by inverse PCR (assuming you know where the exon starts and stops of course).

Make a 30-35microL PCR (depends how low is the amplicon concentration that you get) , run some in a gel just to know if there is amplification then reamplify some of the PCR product (begin with 1-2 microL and increase if necessary). Use proofreading hot star in both reactions. Or make a 50 microL reaction make a very thick gel and run each sample in several wells and cut what you need.

I would go with phage434's suggestions. This can help.