dna sequencing amount - (Sep/19/2005 )

hi everyone,

i just want to ask how much is the minimum amount of pcr product to be submitted for sequencing? If i only have a 20 ul pcr product and check it in agrose, how much should i load in the gel and how much must i send for sequencing? thank you

hi
for sequencing i start from 1 to 2.4 µg using the amersham dye sequencing kit.

50-100 ng of PCR products are sufficient with the DYEnamic protocol

Seb_

hi thanks for the info. i just want to ask if that amount, shoudl there be no bands in agarose using only 5 ul of pcr product then i can try still having it sequenced? thanks

I would strongly recommend that you not try sequencing a PCR product without gel purification.
So, you should run all of your product on the gel, cut out the band, gel purify, and submit for sequencing. You need only about 20 ng for sequencing, but it has to be purified away from the excess primers and dNTPs of your PCR reaction, and from any primer-dimers which may have formed.
It is *not* necessary to run the purified DNA on a gel again -- in some cases, the bands may be invisible when you do this, and the sequencing can still work out fine. The limit for EtBr detection is about 5-10 ng of DNA on an agarose gel. A narrow well and destaining of the gel will increase sensitivity.