SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Sample Prep / Library Generation (http://seqanswers.com/forums/forumdisplay.php?f=25)
-   -   Blue Pippen problems (http://seqanswers.com/forums/showthread.php?t=70289)

kgoglin 07-14-2016 04:14 PM

Blue Pippen problems
 
Is anyone else having problems with Blue Pippen's accuracy? Our error % from all our control DNAs tests range from 3-13%.

One Example:
Tight Setting(250bp)
Bioanalyzer average size (bp) (282bp)
12.8% error

What is an acceptable error rate for the Blue Pippen?

We have been using a 20bp offset for our samples (both amplicon and sheared gDNA) and BP control DNA. (So for a 390bp extraction, we use the 370bp tight setting.) This has been working for a while, but not anymore. We contacted Sage Science, who has been helping us a little, but we are still running through loops. Sage says our BP instrument log files are great and that it could be a cassette issue. They also think that since we are using PCR product, we may have single-stranded DNA that may be interfering with how we visualize peaks on the Bioanalzyer. This may be true but our sheared gDNA does not size accurately either.

Sage Science suggests that, for every new sample type, we run a test run to determine the best setting. Does anyone do this?

What other problems have you seen with the Blue Pippen? Is there a better alternative?

nucacidhunter 07-14-2016 06:44 PM

One has to take into account size distribution CV, reproducibility% and accuracy% for each Cassette type. Loading quantity, structure of DNA (blunt ended, Y shape, staggered ends) also can affect the size range distribution. On top of all of these also BA has its own specifications and deviation from them also will affect the sizing.

Your example of tight setting at 370bp will collect fragments in 337-403bp. If you use range setting of 350-390 it will not collect 370bp as that setting is narrower than tightest setting possible for which the software gives a narrow warning.

I agree with Sage explanation and their platform is much better than another one that I have tried.

kerplunk412 07-18-2016 11:32 AM

I know that to select small RNA libraries of ~150 bp and exclude adapter-dimer at ~130 bp you must use settings way different from expected (115 bp-170 bp collection range), so I am not surprised that a little optimization may be necessary.

Regarding a better alternative, the only other thing I have tried is the E-gel, and Pippin is so much better it isn't even a comparison. I think the Caliper LabChip is probably a better comparison but I haven't had a chance to try it. I did find this Googling a little though.

nucacidhunter 07-19-2016 12:13 AM

I had a similar result to the linked paper in kerplunk412 post comparing Pippin and LabChip.

kgoglin 07-22-2016 10:38 AM

Thank you for responding :)

Sage Science said that they like to see less than 10% error from the control DNA. This seems pretty high to me but if that's the case, then I guess we will have to test more than one setting for each sample type.


All times are GMT -8. The time now is 05:42 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.