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
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
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