[aahardikar]

We use the bioanalyzer as well for all runs and AmPure for size selection. How do you get the e-gels to work?

[lisazgreen]

Attached is experiment I performed last year comparing E-Gel® SizeSelect™ to PippinPrep (SAGE Science) for the size selection step in Ion LC, prior to library amplification.

SizeSelect Run Conditions:

50 bp DNA Ladder Cat#10416-014

Run SizeSelect™ 2% (program 8)

Time for 185bp DNA target to Reach Reference= 15:04 min
Time for 185bp DNA target to Reach Collection Well= 18:00 min

[thomasblomquist]

We use the bioanalyzer as well for all runs and AmPure for size selection. How do you get the e-gels to work?

You put the sample you want gel-purified in the starting well. Press the "Go" button. Then pipette out your band size of interest ~10-15 minutes later (depending on size required). Cost is $13 per gel. And you can run up to 8 library purifications at a time. About $1-2 per library.

I have been using the e-gel for about 4 months now, and have received very consistent library preps from them. Yes, the yield is low nM, but compared to what you need for Ion Torrent Library prep, this is many-fold magnitude more than what you will need for down stream applications. Highly recommend the e-gel for cost, time and reproducibility for NGS library prep.

[aahardikar]

Thanks for your reply....
Do believe it is as simple as you explain. Do you use the elution buffer for e-gels to elute the bands of interest? Any cross-contamination issues with loading?

[thomasblomquist]

Thanks for your reply....
Do believe it is as simple as you explain. Do you use the elution buffer for e-gels to elute the bands of interest? Any cross-contamination issues with loading?

I haven't had any cross-contamination in my controls as of yet. RNA-ase free H2O is the elution buffer. Simple enough to swap out for some sort of tris edta buffer if you wish. It's a very simple system. Google: Invitrogen e-gel size select