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Forum: Sample Prep / Library Generation 01-09-2012, 12:51 PM
Replies: 13
Views: 4,248
Posted By DMO
I would repurify the samples, sounds like maybe...

I would repurify the samples, sounds like maybe you have a contaminant in the sample screwing with the run.
Forum: Sample Prep / Library Generation 01-09-2012, 11:38 AM
Replies: 13
Views: 4,248
Posted By DMO
In addition to that they need regular scrubbing....

In addition to that they need regular scrubbing. We scrub ours with a sterile toothbrush first with MilliQ water, then isoproponal, and again with MilliQ. you then need to let them dry out very well...
Forum: Sample Prep / Library Generation 01-09-2012, 11:30 AM
Replies: 13
Views: 4,248
Posted By DMO
Do you scrub the electrodes regularly?

Do you scrub the electrodes regularly?
Forum: Sample Prep / Library Generation 01-06-2012, 10:00 AM
Replies: 11
Views: 4,194
Posted By DMO
I would extract the DNA from those and amp those....

I would extract the DNA from those and amp those. I think you would be fine sequencing what you have, but you would have to scale back your loading concentration on your run a bit. So, if you...
Forum: Sample Prep / Library Generation 01-06-2012, 09:55 AM
Replies: 11
Views: 4,194
Posted By DMO
You certainly could since you are getting plently...

You certainly could since you are getting plently of material, but based on these parameters I am less likely to jump to the overamplification conclusion. What size did you cut from your gel?
Forum: Illumina/Solexa 01-06-2012, 09:45 AM
Replies: 15
Views: 4,930
Posted By DMO
But how much of your material was in the size...

But how much of your material was in the size range that is getting selected? Thats why I was asking about whether you manually integrated your traces at any point.
Forum: Sample Prep / Library Generation 01-06-2012, 09:30 AM
Replies: 11
Views: 4,194
Posted By DMO
I see smaller peaks on at least 2 of your traces...

I see smaller peaks on at least 2 of your traces which may indicate the samples were quite overamplified.

Can you answer a few more questions?

What is you shearing protocol?

What amount of...
Forum: Sample Prep / Library Generation 01-06-2012, 08:45 AM
Replies: 11
Views: 4,194
Posted By DMO
Do you have a BioA trace post-sonication?

Do you have a BioA trace post-sonication?
Forum: Illumina/Solexa 01-05-2012, 04:24 PM
Replies: 15
Views: 4,930
Posted By DMO
Did you do manual integration of your bioanalyzer...

Did you do manual integration of your bioanalyzer run to see how much of your 25ng is in the 200-400bp range?
Forum: Sample Prep / Library Generation 01-05-2012, 04:17 PM
Replies: 11
Views: 4,194
Posted By DMO
What was your ave bp size post sonication? ...

What was your ave bp size post sonication?

Do you have a picture of your gel or BioA QC of the final product?

Whether or not you should move into sequencing will depend upon whether this was...
Forum: Sample Prep / Library Generation 01-05-2012, 04:14 PM
Replies: 13
Views: 4,248
Posted By DMO
Are you denaturing your ladder and samples for a...

Are you denaturing your ladder and samples for a few minutes before loading on the chip?
Forum: Illumina/Solexa 01-05-2012, 03:51 PM
Replies: 15
Views: 4,930
Posted By DMO
I would recommend prepping manually as the...

I would recommend prepping manually as the SPRI-TE loses a LOT of DNA in its prep. I would only recommend using the SPRI-TE with 100ng+ of starting material. I regularly prep with 10ng (sometimes...
Forum: Sample Prep / Library Generation 02-18-2011, 01:03 PM
Replies: 23
Views: 14,386
Posted By DMO
Cluster Density

When you get 120million reads/lane, what does your cluster density look like?
Forum: Illumina/Solexa 01-28-2011, 09:06 AM
Replies: 2
Views: 4,061
Posted By DMO
Its proprietary, I have asked. If anyone has it,...

Its proprietary, I have asked. If anyone has it, I would be very interested in knowing it as well.
Forum: Sample Prep / Library Generation 01-27-2011, 06:45 PM
Replies: 23
Views: 14,386
Posted By DMO
Yes. The insert size for the HMW buffer with...

Yes. The insert size for the HMW buffer with Nextera is much larger than the target insert of 175bp for the Agilent SureSelect protocol. This is going to affect the efficiency of the hyb and the...
Forum: Sample Prep / Library Generation 01-25-2011, 08:28 AM
Replies: 23
Views: 14,386
Posted By DMO
We have tried it out as well. I agree with...

We have tried it out as well. I agree with NextGen about the prep, it is rather long and tedious. We are still sequencing our first batch, so not sure how the data looks yet.
Forum: Illumina/Solexa 01-24-2011, 06:56 PM
Replies: 4
Views: 3,230
Posted By DMO
Well, you still get the same average number of...

Well, you still get the same average number of reads per lane, just divvied up among the respective samples in the lane. So you just need to figure out how many reads you need for each sample and...
Forum: Illumina/Solexa 01-24-2011, 12:43 PM
Replies: 4
Views: 3,230
Posted By DMO
You can index many more than 12. We have designed...

You can index many more than 12. We have designed primers for 96 indexes and have been using them for the last 6 months or so with very good results (nice even representation across all 96). For many...
Forum: Sample Prep / Library Generation 01-18-2011, 09:35 PM
Replies: 5
Views: 3,537
Posted By DMO
We regularly do all of our cleanups and size...

We regularly do all of our cleanups and size selections by Ampure XP beads. The only tricky part is account for the PEG in your ligation buffer if you are going straight into size selection.
...
Forum: Sample Prep / Library Generation 01-17-2011, 07:27 AM
Replies: 9
Views: 6,839
Posted By DMO
Having intact DNA is important in order to get...

Having intact DNA is important in order to get even shearing across the genome, and thereby more even representation of the genome in your final sequence data. If you have degradation you need to...
Forum: Sample Prep / Library Generation 01-14-2011, 09:06 AM
Replies: 9
Views: 6,839
Posted By DMO
Buffer = TE (or water) Quantity = 3ug ( you can...

Buffer = TE (or water)
Quantity = 3ug ( you can get away with less than this if you need)
260/280 = >1.7
260/230 = >2
Forum: Sample Prep / Library Generation 12-15-2010, 09:26 PM
Replies: 7
Views: 11,643
Posted By DMO
600bp. You can go above this successfully to...

600bp. You can go above this successfully to about 800 or 1000bp but you need to seriously reduce your loading concentration otherwise your clusters begin to overlap due to the length of your...
Forum: RNA Sequencing 12-10-2010, 09:27 AM
Replies: 9
Views: 6,637
Posted By DMO
Well for large projects using automation DIY kits...

Well for large projects using automation DIY kits are always going to be the way to go.
Forum: Sample Prep / Library Generation 12-10-2010, 09:23 AM
Replies: 3
Views: 2,845
Posted By DMO
Well if the primer concentration is too low then...

Well if the primer concentration is too low then you would start to see a tail or a second larger peak. It doesn't sound like you should be running out of primer though, unless your quantifications...
Forum: Sample Prep / Library Generation 12-10-2010, 09:10 AM
Replies: 3
Views: 2,845
Posted By DMO
What primer concentration are you using?

What primer concentration are you using?
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