primer dimer

[Guest]

Hi everyone,
We have to do a 454 GS junior run but amplimers coming up from our two steps PCR have a visible primer dimer. Do you think this will influence the quality of the run (more short reads, etc) or the AMpure purification is sufficient to get rid of all dimers?
Thank you.

[jdelton]

You may need to do multiple ampure clean-ups to get rid of the dimers. (we occasionally do 2 ampure and a gel slice purification).

[Benh]

Hi jdelton,

Do you mind sharing what's your typical recovery from ampure and gel purification?

When we follow Roche's clean up protocol our recovery is super below like under 10% (even <1%). It becomes not practical to perform clean up when you almost end up with nothing.

Thanks!

[jdelton]

We actually modified Roche's clean up for our ampure. We do 70% of the volume (more or less depending on the ampure calibration which we do with out sizing solution) of the pool - Pool=100ul ampure = 70ul. This clean up is eluted in 100ul and started again. Our final elution is in 30ul. Overall I couldn't tell you what our percent recovery is, but it's enough that it is clearly able to be visualized on an agarose gel and using a bioanalyzer. And more than enough to dilute into emPCR.

[UofG]

We actually modified Roche's clean up for our ampure. We do 70% of the volume (more or less depending on the ampure calibration which we do with out sizing solution) of the pool - Pool=100ul ampure = 70ul. This clean up is eluted in 100ul and started again. Our final elution is in 30ul. Overall I couldn't tell you what our percent recovery is, but it's enough that it is clearly able to be visualized on an agarose gel and using a bioanalyzer. And more than enough to dilute into emPCR.

Do you omit the Sizing Sizing solution completely when performing the AMpure clean up? I have had mixed results using the Sizing Solution, and often wondered if I'd be better off without it.

[jdelton]

Yes we omitted it completely. It was a pain, expensive, and the whole purpose was to have consistent results, yet you still have to calibrate it every time. Plus it didn't flow into our workflow easily.