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Forum: Illumina/Solexa 09-30-2013, 07:54 AM
Replies: 3
Views: 2,902
Posted By Isequencestuff
what MiSeq kit (flowcell) are you using? I assume...

what MiSeq kit (flowcell) are you using? I assume you're using the V2 kit since you're aiming to load 12 pM of total sample. If your quantitation method is accurate, aiming to load 12 pM should give...
Forum: Sample Prep / Library Generation 09-26-2013, 01:13 PM
Replies: 5
Views: 2,714
Posted By Isequencestuff
We use Enzymatics VeraSeq 2.0 HiFi "ultra pure"...

We use Enzymatics VeraSeq 2.0 HiFi "ultra pure" Polymerase. Our negative controls are always blank. Its a seems to be a pretty pure enzyme although it's not a hot start, for that feature I'd go with...
Forum: Sample Prep / Library Generation 09-09-2013, 11:48 AM
Replies: 1
Views: 2,552
Posted By Isequencestuff
Maybe looking at this thread might give you some...

Maybe looking at this thread might give you some insight?

http://seqanswers.com/forums/showthread.php?t=2003
Forum: Illumina/Solexa 06-20-2013, 08:08 AM
Replies: 75
Views: 33,296
Posted By Isequencestuff
Good point. I think trying larger volumes is...

Good point. I think trying larger volumes is worth investigating.
Forum: Illumina/Solexa 06-20-2013, 08:04 AM
Replies: 75
Views: 33,296
Posted By Isequencestuff
qPCR Quantification

The different bars denote individual libraries, and the colors denote a different library preparation. I would disregard all of the colors and different bars, as these libraries were prepped with...
Forum: Illumina/Solexa 06-20-2013, 05:51 AM
Replies: 75
Views: 33,296
Posted By Isequencestuff
Kapa qPCR help

Thank for all the qPCR insights.

Our lab is having some trouble with running the Kapa SYBR qPCR kit and getting reproducible data. We also use the KAPA illumina standards. It could be an obvious...
Forum: Service Providers 04-18-2013, 06:29 AM
Replies: 162
Views: 176,448
Posted By Isequencestuff
University of Texas at Austin Illumina, 454...

University of Texas at Austin Illumina, 454 https://wikis.utexas.edu/display/GSAF/Home+Page
Forum: Illumina/Solexa 02-13-2013, 05:06 AM
Replies: 13
Views: 3,860
Posted By Isequencestuff
we use the dsDNA Qubit kit (or Picogreen) to...

we use the dsDNA Qubit kit (or Picogreen) to quantitate before and after Covaris shearing. We believe that this is the most accurate reading as previously compared to the nanodrop 1000 & Bioanalyzer....
Forum: Sample Prep / Library Generation 01-28-2013, 06:22 AM
Replies: 46
Views: 23,471
Posted By Isequencestuff
For Illumina LC we currently use Invitrogen...

For Illumina LC we currently use Invitrogen E-gels with a modified protocol for Size Selection: After one post ligation Ampure XP purification we elute in 42.5 ul TE Light and recover 40 ul per...
Forum: Sample Prep / Library Generation 01-24-2013, 09:03 AM
Replies: 5
Views: 2,601
Posted By Isequencestuff
What magnet are you using? It seems like your...

What magnet are you using? It seems like your volume might be a little high for efficient bead binding, hence causing the cloudiness. Could you pool, then aliquot out into smaller volumes?
Forum: Sample Prep / Library Generation 01-04-2013, 04:38 AM
Replies: 9
Views: 7,655
Posted By Isequencestuff
Have you tried titrating below 0.7X volume AMPure...

Have you tried titrating below 0.7X volume AMPure beads?
Forum: Illumina/Solexa 12-20-2012, 06:41 AM
Replies: 0
Views: 1,114
Posted By Isequencestuff
Smile Non-sequencing GC Bias

Hi There,

We're trying to recreate the following non-sequencing approach to evaluating GC bias with a few different polymerases. Does anyone have any experience doing so?



Analyzing and...
Forum: Sample Prep / Library Generation 12-17-2012, 06:31 AM
Replies: 1
Views: 3,243
Posted By Isequencestuff
Bioanalyzer DNA Smear Analysis

Hello,

Does anyone know if smear analysis will ever give a 100% area measurement? I'm trying to figure out my standard error upon detecting two regions; first region starting right after the first...
Forum: Sample Prep / Library Generation 12-13-2012, 09:04 AM
Replies: 49
Views: 73,043
Posted By Isequencestuff
We've had experiences with both: completely dry...

We've had experiences with both: completely dry cracking beads, and also still moist. We can't seem to see a difference in yield between the two using AMPure XP (we haven't used regular AMPure). We...
Forum: Introductions 12-11-2012, 11:27 AM
Replies: 0
Views: 872
Posted By Isequencestuff
Smile Hello Everyone!

Hey there fellow NextGens,

My name's Michael and I'm from New York. We're working on low throughput and high throughput sequencing projects for Illumina platform utilizing HiSeq. We've also go a...
Forum: Illumina/Solexa 12-03-2012, 11:39 AM
Replies: 6
Views: 5,366
Posted By Isequencestuff
We've tried Enzymatics' "ultra-pure ligase"....

We've tried Enzymatics' "ultra-pure ligase". Compared to a lot of other ligases on the market there does seem to be a significant increase in ligated fragments as determined by qPCR pre and post...
Forum: Sample Prep / Library Generation 11-27-2012, 01:37 PM
Replies: 7
Views: 1,483
Posted By Isequencestuff
Ah I see, thanks for the recommendations. I'll...

Ah I see, thanks for the recommendations. I'll give them a try.
Forum: Ion Torrent 11-27-2012, 01:31 PM
Replies: 0
Views: 1,745
Posted By Isequencestuff
Measure Nick Translation efficiency?

Hey guys,

In the Ion Torrent library construction workflow, is there any way to measure the efficiency of the nick translation step? I'm not sure how to measure this step of library preparation....
Forum: Sample Prep / Library Generation 11-27-2012, 12:38 PM
Replies: 7
Views: 1,483
Posted By Isequencestuff
Thanks for the magnet and tube recommendations....

Thanks for the magnet and tube recommendations. I'll look into both of those magnets as well as my surgical pipetting skills when it comes to End Repair purification. PCR strips seem good for low...
Forum: Sample Prep / Library Generation 11-27-2012, 11:12 AM
Replies: 7
Views: 1,483
Posted By Isequencestuff
Alright, 0.2 mL PCR strips sound ideal since...

Alright, 0.2 mL PCR strips sound ideal since incubation temperatures are vital. Can you recommend a magnet that has worked best for you? If you're using AMPure beads to purify, can you fit the entire...
Forum: Sample Prep / Library Generation 11-27-2012, 05:36 AM
Replies: 7
Views: 1,483
Posted By Isequencestuff
Library Construction Format

Hey Guys,

I'm rather new to library construction & sequencing and am unsure of a few things. A little background: I'm using the Truseq sample preparation kit for library workflow, and my colleague...
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