Poor Enrichment, expired/thawed reagents, maintenance washes, and cDNA

[Anthony.287]

Hi everybody! I’ve been working with the 454 FLX Titanium for about 8 months now, and I have some pretty specific questions that I’m having a hard time finding answers to, so I’m hoping that some of you can help me out.

Enrichment numbers – I completed an emPCR titration (SV, 2 to 16 cpb) run this week, with a cDNA library, and had very low enrichment numbers (around 100,000 beads, or less than 1%) and have no idea why. The Rapid Library adaptors seemed to be ligated, based on the fluorometer readings, and I had good bead recovery (around 70%), but next to no enriched beads. I plan on trying it again this week. Any ideas?

Freezing reagents/expiration date – I know the manuals say not to refreeze the sequencing reagents, but I had pulled the 50mL tubes out of the freezer and placed them at 4 degrees overnight to thaw, in anticipation of performing a sequencing run, but then my enrichment numbers were so low that I didn’t bother to sequence them. I placed the sleeve with the 50mL tubes back into the freezer (after being in the fridge for ~24 hours) in hopes that it would be okay for a future run. Does anyone know if these reagents will be alright, or should I toss the whole bunch? Also, we have a set of sequencing reagents that expired in April of this year. Does anyone have experience using sequencing reagents past the expiration date?

Home-made maintenance washes – I found on this forum the concentration of bleach used for the maintenance washes (the Tween is marked on the side of the tube), and am wondering if anyone has experience using “home-made” maintenance washes. I’m sure Roche doesn’t recommend this, but does anyone know if this would violate any service contracts? I’d really like to save the lab some money, if at all possible.

Fragmenting cDNA – finally, the cDNA library I mentioned above was created with the protocol in the Roche cDNA Synthesis System, NOT the FLX Titanium cDNA Protocol (I couldn’t get a high enough/consistent yield from the FLX protocol) using the Random primer. The fragments were mainly between 550-1500bp (based on the Bioanalyzer data), so I didn’t fragment or nebulize them further. Any recommendations for cDNA fragments this size?

Thank you all in advance for any and all advice you can provide. I’ve been reading these forums pretty extensively for some time now, and have learned a great deal. Hopefully I’ll be able to contribute sometime!!

Thanks again!
Anthony

[ajthomas]

Enrichment numbers – I completed an emPCR titration (SV, 2 to 16 cpb) run this week, with a cDNA library, and had very low enrichment numbers (around 100,000 beads, or less than 1%) and have no idea why. The Rapid Library adaptors seemed to be ligated, based on the fluorometer readings, and I had good bead recovery (around 70%), but next to no enriched beads. I plan on trying it again this week. Any ideas?

Did you get the same number of beads from each reaction, or did you get different numbers that corresponded with the amount of library used in each one? If you got the same number from each titration, I suspect you actually didn't get anything and the emPCR completely failed. If that's the case, you may have the same issue we've been having lately in that the last several libraries we have made (by three different people) all looked good based on quantification and the Bioanalyzer, but completely failed in emPCR. We don't know for sure, but we suspect that for some reason the adapters were only ligated on one strand and not the other (perhaps due to bad T4 PNK?). We got a new library prep kit and we should know tomorrow whether that solved the problem.

If you did get more beads from the higher amounts of DNA, then it probably worked and the library was either not correctly quantified, or the ligation just didn't work very well. In that case, you may be okay just using more DNA until you get a good enrichment percentage.

[Anthony.287]

No, there was no correlation between the amount of library and the number of beads recovered. I thought that there may have been, but no.
In trying to troubleshoot the emPCR, I found some discrepancies in the concentration calculations, corrected those, and attempted a single tube emPCR reaction to test whether or not that fixed it. It did not.
I think I may start over with a new library prep.

[Christina85]

Did you get the same number of beads from each reaction, or did you get different numbers that corresponded with the amount of library used in each one? If you got the same number from each titration, I suspect you actually didn't get anything and the emPCR completely failed.

Hi!

I have performed emulsion titration with 2 x 10^6 Capture Beads per emulsion tube and 4 x 10^6 Capture Beads per emulsion tube, but in some cases the 2 cpbs had a higher enrichment value than the 4 cpbs (for example 46,85% over 41,45%) and in some other cases it was the opposite (for example 36,84% over 40,23%). Do you think that the emPCR has failed?

I have posted all the details on "Calculation of % Bead enrichment (454 FLX Titanium)": http://seqanswers.com/forums/showthread.php?t=18588

Thank you!!