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Forum: Illumina/Solexa 01-10-2018, 05:58 PM
Replies: 2
Views: 866
Posted By kgoglin
Bioanalyzer and qPCR concentrations differ with large libraries

We have a theory that large libraries, specifically those with fragments trailing off to >2kb, do not quantitate accurately from Bioanalzyer. We think this because qPCR concentrations do not...
Forum: Sample Prep / Library Generation 02-02-2017, 01:38 PM
Replies: 1
Views: 753
Posted By kgoglin
mRNA jaggeed peaks in profiles on Bioanalzyer

An mRNA profile on Bioanalyzer can take slightly unusual forms. Rarely do we see a perfect clean hump (see attached). Sometimes we get a hump with many jagged sharp peaks and within the same sample...
Forum: Sample Prep / Library Generation 02-02-2017, 01:25 PM
Replies: 2
Views: 1,130
Posted By kgoglin
Yeah. I guess we could just make our own. ...

Yeah. I guess we could just make our own.

Thanks!
Forum: Sample Prep / Library Generation 01-30-2017, 02:17 PM
Replies: 2
Views: 1,130
Posted By kgoglin
PCR index primers for Illumina RNA-Seq

We currently use Illumina's ScriptSeq index primers for PCR amplifying cDNA produced by Wafergen's PrepX mRNA protocol. The ScriptSeq index primers only allow for 48 indexes. Are there any other...
Forum: Sample Prep / Library Generation 01-13-2017, 11:26 AM
Replies: 6
Views: 4,151
Posted By kgoglin
I have used Aline beads for normalization in the...

I have used Aline beads for normalization in the past with NEBNext libraries. The beads worked well except the final normalized concentration was variable when using different lots. I think Aline...
Forum: Core Facilities 12-12-2016, 01:10 PM
Replies: 3
Views: 2,189
Posted By kgoglin
Thanks for your response. We are not sure what...

Thanks for your response. We are not sure what the downstream sequencing would look like if we performed library prep on one of the DNA contaminated samples. Since the Bioanalzyer profile looked...
Forum: Core Facilities 12-12-2016, 11:54 AM
Replies: 3
Views: 2,189
Posted By kgoglin
RNA QC: DNA contamination bad for rRNA depletion/mRNA capture

DNA contamination in an RNA sample can be detrimental to the rRNA depletion or mRNA capture assay prior to RNASeq library prep. We currently use the Bioanalyzer for RNA QC but it is not satisfactory...
Forum: Core Facilities 08-31-2016, 05:39 PM
Replies: 7
Views: 2,931
Posted By kgoglin
This is all great information and on all...

This is all great information and on all different levels. Thank you!
Forum: Core Facilities 08-25-2016, 09:56 AM
Replies: 7
Views: 2,931
Posted By kgoglin
How to increase sample submission to core

As a sequencing core facility, we encourage those in our institution to submit their samples to us. We perform DNA/RNA extractions, library prep, and sequencing on Illumina NextSeq and MiSeq...
Forum: Sample Prep / Library Generation 07-29-2016, 08:56 AM
Replies: 2
Views: 1,070
Posted By kgoglin
The two peaks I'm referring to are not the 16S...

The two peaks I'm referring to are not the 16S and 23S. If you open the pdf file and look at the 30 hour samples, you will see what I am referring to.

We processed these samples through...
Forum: Sample Prep / Library Generation 07-22-2016, 11:57 AM
Replies: 2
Views: 1,070
Posted By kgoglin
Strange double-peak in Agilent RNA profile

We are learning to pay attention to extraneous peaks seen in RNA profiles from the Agilent RNA nano chip. If we see any sort of tiny peaks around the 23S peak, we automatically assume DNA...
Forum: Sample Prep / Library Generation 07-22-2016, 11:38 AM
Replies: 4
Views: 1,177
Posted By kgoglin
Thank you for responding :) Sage Science...

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...
Forum: Sample Prep / Library Generation 07-14-2016, 05:14 PM
Replies: 4
Views: 1,177
Posted By kgoglin
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)...
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