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Forum: General 08-06-2013, 11:04 AM
Replies: 0
Views: 1,626
Posted By OnUrMark
Changing Color of Bases in BioEdit Chromatograms

Greetings!
I want to change the colors used for C and/or G in the chromatograms of BioEdit. I found code info for changing the table view. Does anyone know if we can access the code file for the...
Forum: Sample Prep / Library Generation 05-20-2013, 09:32 AM
Replies: 3
Views: 2,945
Posted By OnUrMark
Hi Krobinson, Thanks for your advice! I have...

Hi Krobinson,

Thanks for your advice! I have access to a Qubit for DNA quantitation, and I would like to clean the minipreps I have in hand without amplification.

It sounds like the main thing...
Forum: Sample Prep / Library Generation 05-17-2013, 01:46 PM
Replies: 3
Views: 2,945
Posted By OnUrMark
How to prep BACs for Nextera XT DNA kit?

Greetings All,

I need help working with BACs. I need DNA from 13 BACs for use with the Nextera XT DNA kit to be sequenced on the Illumina MiSeq. I am totally totally new to working with BACs....
Forum: Illumina/Solexa 12-23-2011, 12:41 PM
Replies: 45
Views: 26,406
Posted By OnUrMark
We tried this suggestion last week (slow ramp...

We tried this suggestion last week (slow ramp speed on the last cycle). Our bubble product was also greatly increased, but our concentrations determined by KAPA qPCR were the same as our previous...
Forum: RNA Sequencing 11-15-2011, 11:24 AM
Replies: 1
Views: 2,534
Posted By OnUrMark
TruSeq RNA In-Line Control Sequences

Greetings,

I have recently prepared RNA libraries using the TruSeq RNA kit and the in-line controls. Does anyone have the TruSeq RNA in-line control sequences?

Thanks!
Forum: Sample Prep / Library Generation 09-27-2011, 09:42 AM
Replies: 19
Views: 8,348
Posted By OnUrMark
Thanks for the advice, but I have the TruSeq RNA...

Thanks for the advice, but I have the TruSeq RNA kit and all necessary consumables in hand. I need to learn how to make this kit work well for us.
Forum: Sample Prep / Library Generation 09-27-2011, 09:29 AM
Replies: 3
Views: 3,510
Posted By OnUrMark
Hi RNAseqer, I am guessing you mean a 1x...

Hi RNAseqer,

I am guessing you mean a 1x ratio will recover everything >~100bp. I am concerned if this is true because adapter dimers must be removed, and they are ~120bp.

Thanks,

OnUrMark
Forum: Sample Prep / Library Generation 09-26-2011, 12:35 PM
Replies: 3
Views: 3,510
Posted By OnUrMark
TruSeq RNA initial concentration and Ampure beads

Greetings!

I am getting ready to use the TruSeq RNA kit for the first time. I noticed the concentration of Ampure beads to sample decreases as you progress through the steps to prepare an RNASeq...
Forum: Sample Prep / Library Generation 09-26-2011, 12:25 PM
Replies: 19
Views: 8,348
Posted By OnUrMark
Hi Phillip, Did you get to the bottom of the...

Hi Phillip,

Did you get to the bottom of the fragmentation time and median insert length issue? I spoke to a lab that uses 6 seconds, but the only person in the lab did not know what size fragment...
Forum: Sample Prep / Library Generation 06-08-2011, 11:10 AM
Replies: 3
Views: 3,157
Posted By OnUrMark
samples amplify during PCR Enrich but don't sequence

Hi NGS,

I am using a DIY method, and we would really like to solve the problem so that we can use the data collected thus far. If I change methods, there will be a different bias, and this will...
Forum: Sample Prep / Library Generation 06-07-2011, 05:07 PM
Replies: 3
Views: 3,157
Posted By OnUrMark
Hi Cybog337, I know this is an older...

Hi Cybog337,

I know this is an older thread, but I am having a similar problem. Are you using a do-it-yourself method?

Thanks
Forum: 454 Pyrosequencing 05-04-2011, 12:22 PM
Replies: 9
Views: 5,014
Posted By OnUrMark
Hi Tony, Thanks for sharing your gel extraction...

Hi Tony, Thanks for sharing your gel extraction protocol. I have not been leaving more than one lane between samples and sometime the ladder is in the adjacent lane. I am surprised by the high...
Forum: 454 Pyrosequencing 04-21-2011, 12:35 PM
Replies: 9
Views: 5,014
Posted By OnUrMark
Hi Tony, would you mind sharing your gel...

Hi Tony, would you mind sharing your gel extraction technique? I believe there may be contamination from our final gel extraction that is inhibiting the sequencing reaction. The bioanalyzer shows...
Forum: Sample Prep / Library Generation 04-21-2011, 11:22 AM
Replies: 11
Views: 6,221
Posted By OnUrMark
Hello kshankar, I too would like to know why...

Hello kshankar, I too would like to know why indexed libraries may produce lower cluster densities. How many samples per lane are you indexing?

Also, I still believe there are some contaminants...
Forum: Sample Prep / Library Generation 03-31-2011, 05:44 PM
Replies: 11
Views: 6,221
Posted By OnUrMark
Thanks very much for your suggestion. Although we...

Thanks very much for your suggestion. Although we don't use the 3 primer-based barcoding method, it may be informative to run pre gel extracted product on the bioanalyzer.

We ligate barcoded...
Forum: Sample Prep / Library Generation 03-29-2011, 05:24 PM
Replies: 11
Views: 6,221
Posted By OnUrMark
Yes. We PCR enrich the 350bp cDNA 12-18 cycles...

Yes. We PCR enrich the 350bp cDNA 12-18 cycles and then gel extract again. This final sample is checked on the bioanalyzer and the qubit before mixing with other samples for multiplexing and...
Forum: Sample Prep / Library Generation 03-29-2011, 02:17 PM
Replies: 11
Views: 6,221
Posted By OnUrMark
We are using the Qiagen gel extraction kit with...

We are using the Qiagen gel extraction kit with minielute columns. We have changed our gel several times, but presently we are using a 2% NuSieve 3:1 agarose gel (with EtBr) at RT for 80min.
Forum: Sample Prep / Library Generation 03-29-2011, 01:01 PM
Replies: 11
Views: 6,221
Posted By OnUrMark
We use the core facilities here for ngs. I have...

We use the core facilities here for ngs. I have not looked at the high concentration directions from Illumina, but it is my understanding that the core facility dilutes samples so that the NaOH...
Forum: Sample Prep / Library Generation 03-29-2011, 12:04 PM
Replies: 11
Views: 6,221
Posted By OnUrMark
RNAseq - low cluster density - possible inhibitor?

Greetings,
We have been getting low cluster densities, and as we increase the concentration, we are getting less clusters/tile/pM. It looks like there is no way for our samples to reach the optimal...
Forum: RNA Sequencing 02-09-2011, 10:01 AM
Replies: 11
Views: 5,715
Posted By OnUrMark
Ok, thanks. I was misunderstanding what part of...

Ok, thanks. I was misunderstanding what part of the hybrid was ds.
Forum: RNA Sequencing 02-09-2011, 09:49 AM
Replies: 11
Views: 5,715
Posted By OnUrMark
Thank you for your help on this problem. I will...

Thank you for your help on this problem. I will perform gel extraction at RT from now on, and it makes sense that this will alleviate the problem.

I understand Genlyai's description of the...
Forum: RNA Sequencing 02-08-2011, 05:30 PM
Replies: 11
Views: 5,715
Posted By OnUrMark
Thanks for your explanation and ideas. Today I...

Thanks for your explanation and ideas. Today I had several samples denatured before they were run on the bioanalyzer. The 700 bp peaks did disappear for all samples, and all samples had peaks only at...
Forum: RNA Sequencing 01-14-2011, 03:36 PM
Replies: 11
Views: 5,715
Posted By OnUrMark
How to eliminate PE dimers from final product?

Greetings RNA-Seq'ers,

I am preparing samples for RNA-seq, and my final PCR enrichment product contains dimers and trimers of my 350bp fragments (~700bp and ~1000bp). I have gel extracted the PE...
Forum: Sample Prep / Library Generation 01-13-2011, 09:56 AM
Replies: 5
Views: 6,100
Posted By OnUrMark
We are also seeing double peaks in our RNA-seq...

We are also seeing double peaks in our RNA-seq library preps, even though all samples have been gel extracted at 350bp. Does anyone know how it is possible to still have 700 bp fragments after gel...
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