2 replies to this topic

[stagius24]

http://info.med.yale.../tavi/2buff.JPG

Basically, the same pcr reaction was run at 2 different buffer. When using 1.6x PCR product, the rxn yeild more DNA but result in big smearing .. I wondered if this is ok in cloning or not. (Let say there is one band with big smear above and below) . Should I try to gel purify or ignore this smear and proceed to cloning..

Thank you,

[donny]

stagius24, on 07 September 2010 - 01:06 AM, said:

http://info.med.yale.../tavi/2buff.JPG

Basically, the same pcr reaction was run at 2 different buffer. When using 1.6x PCR product, the rxn yeild more DNA but result in big smearing .. I wondered if this is ok in cloning or not. (Let say there is one band with big smear above and below) . Should I try to gel purify or ignore this smear and proceed to cloning..

Thank you,

The "smear" could just be because there's more DNA so they are more obvious than those on the right. If you run less of your product, you probably won't see the smear. I would gel purify for cloning regardless. My 2c.

[pito]

Looks ok for me.

If this "smearing" is a reason not to use the products... I would had to trow away almost 99% of my samples

And yes: you need to do a gel purify and maybe even an ethanolprecipitation for cloning anyway.