We are having trouble sequencing BACs. We use the ABI Big Dye kit. If anyone has suggestions/protocols about how they make it work, I'd appreciate it. We currently have a success rate below 50%.

KWaraska said:

We are having trouble sequencing BACs. We use the ABI Big Dye kit. If anyone has suggestions/protocols about how they make it work, I'd appreciate it. We currently have a success rate below 50%.

By 50% success do you mean that 1/2 of your reads are garbage? Are you doing any QC on your DNA preps?

we have not been because we are a core and have not gotten to that point yet. What would you recommend for QC? How about concentrations of DNA to start with?

I'd reccomend purifying a lot of the BAC template DNA using the Qiagen Mega Prep kit and that you submit your template DNA at a concentration between 800 and 1000ng/ul. It is important that the A260/A280 ratio be 1.8 or greater, so that the purity is not a problem.

It is also desirable to have your primer stock at a concentration of 6uM in sterile ddH20 before you add it to the PCR.