Concatemer formation for RainDance library prep?

[ngseq]

Hi All,

I am trying to make ligated libraries using the 454 Rapid library prep kit for DNA samples PCR amplified from RainDance merged primer/template droplets. After Ampure beads purification, I saw a very intense high- molecular weight smear (concatemer formation?) on Bioanalyzer DNA high sensitivity chip, with size ranging from 1.5kb to 6kb.

Enriched Beads yield is very low after emulsion PCR using these library, even with a 8 DNA/Bead ratio.

This seems to be very specific to RainDance/PCRed DNA as I don't have any problem using fragmented DNA for library prep. I tried to reduce DNA input to as little as 30ng/reaction but it still did not resolve the high molecular smear. I tried new kits and it didn't help either.

Does anyone have similar experience? Any thoughts/input would be highly appreciated.

Thanks so much in advance!

[pmiguel]

Never even seen a RainDance system. But if you are getting linear multimers from PCR, that suggests your primer/template ratio is going too low at some point. Because you describe the smear as "intense", my recommendation would be to cut back on the number of cycles of PCR.

--
Phillip

[Benh]

Hi All,

I am trying to make ligated libraries using the 454 Rapid library prep kit for DNA samples PCR amplified from RainDance merged primer/template droplets. After Ampure beads purification, I saw a very intense high- molecular weight smear (concatemer formation?) on Bioanalyzer DNA high sensitivity chip, with size ranging from 1.5kb to 6kb.

Enriched Beads yield is very low after emulsion PCR using these library, even with a 8 DNA/Bead ratio.

This seems to be very specific to RainDance/PCRed DNA as I don't have any problem using fragmented DNA for library prep. I tried to reduce DNA input to as little as 30ng/reaction but it still did not resolve the high molecular smear. I tried new kits and it didn't help either.

Does anyone have similar experience? Any thoughts/input would be highly appreciated.

Thanks so much in advance!

Hi ngseq, not sure if you have solved your problem yet... but likely the source for the high mw smear is from the fragmented DNA template that got carried over from PCR. In which case gel purification may solve your problem.

[ngseq]

Hi Benh,
Thanks! You are probbaly right and we'll try gel purification next time.