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Forum: Sample Prep / Library Generation 10-29-2015, 03:09 PM
Replies: 9
Views: 2,876
Posted By docphil
Sorry about the late reply...I have been using a...

Sorry about the late reply...I have been using a concentrator column on my pippin elute to do a buffer change into EB. I found that using a qiagen spin column resulted in a significant yield loss...
Forum: Sample Prep / Library Generation 07-29-2015, 11:41 AM
Replies: 9
Views: 2,876
Posted By docphil
Pippin Elute directly into MiSeq Library

Hello, I recently got a PippinHT loaned to me for a demo, and I am using it for final library pool size selection before loading my library onto a MiSEQ. I am wondering if I can use the Elute...
Forum: Illumina/Solexa 05-05-2015, 11:01 AM
Replies: 16
Views: 5,771
Posted By docphil
Yes we have seen a dramatic decrease in R2...

Yes we have seen a dramatic decrease in R2 quality, about a 30-35% decrease in %>Q30 compared to runs from ~6months ago. We routinely use V2 500cycle kits for 2x251 reads. My PI has spoken to a...
Forum: Illumina/Solexa 05-01-2015, 02:47 PM
Replies: 16
Views: 5,771
Posted By docphil
The quality of our MiSEQs runs has been...

The quality of our MiSEQs runs has been consistently decreasing over the last 6 months, especially after the flip for paired end sequencing (read2). I am wondering what specific run metrics come with...
Forum: Sample Prep / Library Generation 02-10-2015, 04:53 PM
Replies: 1
Views: 1,228
Posted By docphil
T7 or or e.coli ligase for TA adapter Ligation.

Has anyone tried using T7 or e.coli ligase instead of T4 ligase for ligating adapters? My lab uses custom adapters for amplicon sequencing on the MiSEQ. We have been having issues with adapter dimers...
Forum: Sample Prep / Library Generation 10-23-2014, 10:55 AM
Replies: 0
Views: 886
Posted By docphil
Ugly gels after switching to 384w plate from 96

Hello,
My lab use's custom primer sets for HLA genotyping using the Illumina MiSEQ. We have optimized our multiplex primer sets for initial amplification using 96 well PCR plates. These reactions...
Forum: Introductions 09-22-2014, 03:19 PM
Replies: 0
Views: 736
Posted By docphil
Hello fellow Pipetters!

Hello,
My name is David and I am new here. I work at Fred Hutch Cancer Research Center in Seattle Washington. Pretty new to working with NGS and currently running Illumina MiSEQ. My lab is...
Forum: Sample Prep / Library Generation 09-22-2014, 03:09 PM
Replies: 11
Views: 5,839
Posted By docphil
My Lab uses AMPure beads at 0.7x to remove...

My Lab uses AMPure beads at 0.7x to remove primer-dimer and any junk below our target after barcoding and final pooling for MiSEQ. We elute into TE and then run the eluted DNA through a QiaQuick spin...
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