SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 17 of 17
Search took 0.00 seconds.
Search: Posts Made By: anna_m
Forum: Sample Prep / Library Generation 12-23-2015, 05:05 AM
Replies: 9
Views: 2,591
Posted By anna_m
I agree it is safe to run as it is. We carry out...

I agree it is safe to run as it is. We carry out size selection using the blue pippin instrument on our TruSeq nano libraries after amplification and the final library is the eluted sample taken from...
Forum: Sample Prep / Library Generation 12-21-2015, 03:04 AM
Replies: 0
Views: 1,302
Posted By anna_m
TruSeq nano library changes?

Hello, have anyone noticed their TruSeq nano libraries having a higher yield and/or giving larger size distributions recently?

The new version of the protocol (June 2015) has a change in the...
Forum: Illumina/Solexa 10-06-2015, 06:12 AM
Replies: 4
Views: 1,882
Posted By anna_m
HiSeq rapid run overclustering

Hello,
On the Illumina website, it says that the optical raw cluster density for PhiX runs is 850K-1000K clusters/mm2. But, what is the functional range of the instrument in this mode? The GC bias...
Forum: Sample Prep / Library Generation 07-06-2015, 12:02 AM
Replies: 3
Views: 1,516
Posted By anna_m
That's really interesting kmcarr. No, we use the...

That's really interesting kmcarr. No, we use the 1.5% cassette because we size-select the 550bp nano libraries at 640-700bp so the 2% range would be too small.

Thanks to you and and nucacidhunter...
Forum: Sample Prep / Library Generation 07-03-2015, 02:20 AM
Replies: 3
Views: 1,516
Posted By anna_m
Blue pippin size selection elution volume

Hello,
Has anyone has tried eluting their TruSeq DNA libraries in a smaller volume than the 40uL stated in the protocol?

We carry out size selection on our TruSeq nano libraries after the PCR...
Forum: Illumina/Solexa 06-22-2015, 12:11 AM
Replies: 5
Views: 1,267
Posted By anna_m
No, we don't re-use plates. We clean the...

No, we don't re-use plates. We clean the plate-holder wells with bleach, water and ethanol on recommendation by Kapa once a month. We don't use water to dilute the libraries, we use EB-tween. We put...
Forum: Illumina/Solexa 06-18-2015, 05:17 AM
Replies: 5
Views: 1,267
Posted By anna_m
We have a minimum cut off of 28 for the cq values...

We have a minimum cut off of 28 for the cq values and we have been getting as low as 18 recently.
Forum: Illumina/Solexa 06-18-2015, 12:36 AM
Replies: 5
Views: 1,267
Posted By anna_m
qPCR issues

Hello, we use Kapa qPCR kits to quantify our libraries before sequencing. Recently, the NTC wells are giving low cq values which means there are contaminants and therefore the data is not reliable....
Forum: Sample Prep / Library Generation 10-30-2014, 07:42 AM
Replies: 5
Views: 3,079
Posted By anna_m
Hello, It's a size selection issue. The...

Hello,
It's a size selection issue. The protocol doesn't need to be altered, just make sure your SP beads are vortexed a lot (I mean a lot!). I usually mix them in between each sample prep as a...
Forum: Sample Prep / Library Generation 10-29-2014, 01:18 AM
Replies: 2
Views: 3,041
Posted By anna_m
NEBNext Ultra-Directional RNA Library Prep Kit

Hello,
Has anyone tried the NEBNext Ultra Directional RNA Library Prep Kit? If so, how were your final libraries? Any feedback on this would be great, thanks.
...
Forum: Sample Prep / Library Generation 04-08-2014, 05:19 AM
Replies: 5
Views: 3,079
Posted By anna_m
Hello, I asked Illumina tech support and they...

Hello, I asked Illumina tech support and they said to try diluting the libraries as the chip looked to be overloaded. I made a 1 in 20 dilution and repeated the chip. An example of a trace is...
Forum: Sample Prep / Library Generation 04-07-2014, 02:55 AM
Replies: 5
Views: 3,079
Posted By anna_m
Larger size TruSeq nano prep

Hello,
I followed the protocol for the 550bp size TruSeq nano library preparation but my final libraries were much larger than the expected 670bp average size (see attached pic).

Has anyone else...
Forum: Sample Prep / Library Generation 10-08-2013, 05:11 AM
Replies: 0
Views: 1,587
Posted By anna_m
Bimodal distribution in TruSeq libraries

Hi,
I made some control TruSeq genomic gel-free libraries using Ecoli and pooled these together equimolar. This pool then underwent blue pippin size selection at 540bp. The plot after duplicate...
Forum: Sample Prep / Library Generation 09-10-2013, 05:54 AM
Replies: 2
Views: 2,537
Posted By anna_m
Extra peak after TruSeq DNA library prep

Hello,
We made library preps following the standard TruSeq DNA library prep method, beginning with 3ug gDNA and took this through the steps shearing-end repair-A tailing-ligation-size selection-PCR....
Forum: Sample Prep / Library Generation 06-17-2013, 05:56 AM
Replies: 7
Views: 3,883
Posted By anna_m
Hi, Thanks for your reply. 2 minutes just...

Hi,
Thanks for your reply. 2 minutes just doesn't seem enough. Is there not any ethanol carryover this way? I keep an eye on the beads and usually 10-15 minutes is fine so that the beads appear matt...
Forum: Sample Prep / Library Generation 06-17-2013, 05:54 AM
Replies: 0
Views: 2,140
Posted By anna_m
Size selection for TruSeq DNA libraries

Hello,
When is the best time for carrying out size selection of TruSeq DNA libraries - pre or post PCR? What kind of adapters are labs generally using?

We've been doing a method of using a paired...
Forum: Sample Prep / Library Generation 06-17-2013, 05:47 AM
Replies: 7
Views: 3,883
Posted By anna_m
Ampure bead clean up

Hello,
How much drying time do you normally allow when using ampure beads XP to clean up your library preps?

I've been using the standard 15 minutes as mentioned in the TruSeq DNA library prep...
Showing results 1 to 17 of 17

 


All times are GMT -8. The time now is 06:08 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO