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Forum: Sample Prep / Library Generation 12-26-2011, 05:53 PM
Replies: 49
Views: 72,614
Posted By edawad
I've just been drying 5 minutes under a hood,...

I've just been drying 5 minutes under a hood, spinning down, and resuspending. Ampure XP has been so efficient regardless that I always have plenty of library (usually 10x more than doing the 2nd gel...
Forum: Illumina/Solexa 12-23-2011, 12:48 PM
Replies: 45
Views: 25,700
Posted By edawad
In our lab, we've either re-PCRd with 4x primer,...

In our lab, we've either re-PCRd with 4x primer, or take the bubble'd product and perform an additional 2 cycles of PCR with extra primer. Both of these methods worked to remove the extra HMW product.
Forum: Illumina/Solexa 08-01-2011, 08:48 AM
Replies: 45
Views: 25,700
Posted By edawad
I did several PCR reactions to test (had enough...

I did several PCR reactions to test (had enough pre-pcr sample to spare). I tried 0.5, 1, 2, and 4 ul of my 10 nm working soln primer with both 15 and 18 cycles, and only using 4 ul got rid of the...
Forum: Illumina/Solexa 08-01-2011, 07:44 AM
Replies: 45
Views: 25,700
Posted By edawad
Thought I'd throw in my experiences with HMW...

Thought I'd throw in my experiences with HMW smears. I was preparing libraries using the NEBnext kit and custom barcode PE adapters. The libraries had a normal peak at 350, then a consistent smear...
Forum: Sample Prep / Library Generation 04-01-2011, 12:44 PM
Replies: 49
Views: 72,614
Posted By edawad
When using Ampure XP beads, the included protocol...

When using Ampure XP beads, the included protocol says to dry for <5 min after the EtOH wash to avoid beads drying out too much and cracking. However most online protocols I see, plus Illumina's...
Forum: Sample Prep / Library Generation 03-16-2011, 02:03 PM
Replies: 1
Views: 1,881
Posted By edawad
Are you thinking of something similar to a...

Are you thinking of something similar to a re-ChIP, where you find what DNA is bound to a complex of both proteins? You can do a normal ChIP with one antibody, then after eluting just ChIP that with...
Forum: Sample Prep / Library Generation 02-23-2011, 04:51 PM
Replies: 2
Views: 3,334
Posted By edawad
Don't worry about nanodropping your DNA after...

Don't worry about nanodropping your DNA after ChIP. The concentration is most likely <10ng/ul given your conditions so if anything you should be using a Qubit. Just proceed with your favorite...
Forum: Epigenetics 02-08-2011, 03:17 PM
Replies: 8
Views: 7,806
Posted By edawad
PE is almost always better for RNA-seq--you gain...

PE is almost always better for RNA-seq--you gain more information about splice junctions etc. It doesn't hurt for ChIP-Seq and may help you better identify enriched binding sites in repetitive...
Forum: Illumina/Solexa 02-02-2011, 02:12 PM
Replies: 45
Views: 25,700
Posted By edawad
upenn_ngs: can you elaborate on your experience...

upenn_ngs: can you elaborate on your experience using the E-gel with the Truseq adapters? How much was the migration affected by using truseq vs old illumina/homemade adapters? Do you do a 2nd E-gel...
Forum: Illumina/Solexa 02-01-2011, 04:23 PM
Replies: 2
Views: 3,704
Posted By edawad
ChIP-Seq with truseq kit?

Hi all,
I'm thinking of switching to the Truseq kit for their indexed primers etc. They have kits for DNA, RNA, and smallRNA. Has anyone heard of or tried using the truseq DNA kit for ChIP-Seq (we...
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