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Forum: Sample Prep / Library Generation 06-11-2018, 01:18 PM
Replies: 5
Views: 1,777
Posted By DNA_Dan
Old post I know, sorry to be late to the party,...

Old post I know, sorry to be late to the party, but if you're still around RickC7, I'd like to see if you made any inroads on the normalization front. It seems like labs doing a lot of samples use...
Forum: Introductions 09-22-2016, 07:35 AM
Replies: 6
Views: 1,429
Posted By DNA_Dan
The only possibility I think NGS can help is if...

The only possibility I think NGS can help is if perhaps you treat the PCR product as a genome and use a low-input library kit to get the adapters on the fragments. If you can do this, then it will...
Forum: 454 Pyrosequencing 09-21-2016, 08:05 PM
Replies: 5
Views: 9,288
Posted By DNA_Dan
How big are your targets? We cover small HIV...

How big are your targets? We cover small HIV regions on a MiSeq 600 cycle kit and can multiplex up to 384 samples in a single run using the Nextera XT adapters.
Forum: Introductions 09-21-2016, 07:59 PM
Replies: 6
Views: 1,429
Posted By DNA_Dan
The best you'll do on an Illumina platform is...

The best you'll do on an Illumina platform is ~600 bp targets on a MiSeq paired-end 2x300 bp kit. It takes time to develop primers for your targets, but once they are made the advantage of NGS is you...
Forum: Sample Prep / Library Generation 03-08-2016, 12:12 PM
Replies: 28
Views: 11,802
Posted By DNA_Dan
We're using an older Applied Biosystems 7900HT.

We're using an older Applied Biosystems 7900HT.
Forum: Sample Prep / Library Generation 03-08-2016, 07:12 AM
Replies: 28
Views: 11,802
Posted By DNA_Dan
Assuming the libraries range across the board,...

Assuming the libraries range across the board, Truseq, Nextera, Kapa Biosystems, Nugen, etc., come from a huge variety of DNA/RNA sources, and have different fragment lengths/profiles - How tight...
Forum: Sample Prep / Library Generation 03-05-2016, 06:20 AM
Replies: 28
Views: 11,802
Posted By DNA_Dan
Old thread but same issues with qPCR. I am...

Old thread but same issues with qPCR. I am surprised that no one is jumping on the ddPCR bandwagon. I guess it just has it's own issues and isn't that popular. Anyone with experience care to comment?...
Forum: Sample Prep / Library Generation 08-03-2015, 09:28 AM
Replies: 4
Views: 3,727
Posted By DNA_Dan
I think this has to do with the mobility of the...

I think this has to do with the mobility of the y-forked adapters. Agarose is more difficult for migration because it's a solid phase, whereas the Bioanalyzer chip's gel matrix is much more fluid. I...
Forum: Illumina/Solexa 11-20-2014, 09:24 AM
Replies: 18
Views: 7,737
Posted By DNA_Dan
Contamination aside, have any of you looked at...

Contamination aside, have any of you looked at the effect of resampling bias from doing so much PCR on a sample? I have a customer looking to do this approach coming from RNA. This means 4 rounds of...
Forum: Illumina/Solexa 11-13-2014, 07:44 PM
Replies: 18
Views: 7,737
Posted By DNA_Dan
As a rule of thumb, we never use the same barcode...

As a rule of thumb, we never use the same barcode combination twice on a plate. It's not like you have the option of running the same barcodes in multiple lanes like you can on a HiSeq.

For us the...
Forum: Sample Prep / Library Generation 11-13-2014, 01:47 PM
Replies: 4
Views: 3,727
Posted By DNA_Dan
Make sure you aren't overloading the chip. The...

Make sure you aren't overloading the chip. The first 3-5 samples look really hot relative to the standard. You could also try a DNA1000 chip since your products are so small.
Forum: Illumina/Solexa 11-13-2014, 01:37 PM
Replies: 18
Views: 7,737
Posted By DNA_Dan
I think Illumina recommends a two-step approach....

I think Illumina recommends a two-step approach. Fusion primers with barcode and sequencing primer locations, then a second PCR with P5/P7 ends being added. If you do it all at once it may get ugly....
Forum: Sample Prep / Library Generation 11-06-2014, 10:13 AM
Replies: 28
Views: 11,802
Posted By DNA_Dan
Anyone have any feedback on the Biorad QX200?...

Anyone have any feedback on the Biorad QX200? Does it work as advertised for NGS quantitation or is this just "smoke and mirrors"? Seems like the next logical step to take.
Forum: Illumina/Solexa 11-05-2014, 09:56 AM
Replies: 50
Views: 37,249
Posted By DNA_Dan
Regarding this comment from a while back - if...

Regarding this comment from a while back - if your target is a PCR amplicon product and the ends are all the same primer sequences, isn't the ONLY way to successfully sequence this is through a...
Forum: Illumina/Solexa 06-18-2013, 08:03 AM
Replies: 75
Views: 33,445
Posted By DNA_Dan
I'm not that qPCR saavy but I get what you're...

I'm not that qPCR saavy but I get what you're saying. I definitely would like to take a look at what you have. Anything that makes this more consistent is a win for everyone on the forums.
Forum: Illumina/Solexa 06-17-2013, 07:09 AM
Replies: 75
Views: 33,445
Posted By DNA_Dan
Does your PCR reaction efficiency change with a...

Does your PCR reaction efficiency change with a more dilute sample?

I've always looked at it as being relative to the standard. As long as the slope is always the same and the standards come off...
Forum: Illumina/Solexa 06-14-2013, 02:18 PM
Replies: 75
Views: 33,445
Posted By DNA_Dan
I'm with GW_OK. A nanadrop really has no business...

I'm with GW_OK. A nanadrop really has no business in NGS as far as I am concerned. It measures everything, whereas the Kapa kit is measuring only "functional" molecules that will actually contribute...
Forum: 454 Pyrosequencing 10-23-2012, 10:10 AM
Replies: 24
Views: 5,927
Posted By DNA_Dan
See Application Brief # 001-2009 Entitled...

See Application Brief # 001-2009 Entitled Unidirectional Sequencing of Amplicon Libraries Using the GS FLX TItanium emPCR Kits. (Lib-L) On the second page, Primer A and Primer B are for fusion...
Forum: 454 Pyrosequencing 10-22-2012, 12:49 PM
Replies: 24
Views: 5,927
Posted By DNA_Dan
This might be a possibility, but the equilibrium...

This might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments...
Forum: Illumina/Solexa 07-30-2012, 09:57 AM
Replies: 75
Views: 33,445
Posted By DNA_Dan
+1 on the Kapa kit. All these methods are...

+1 on the Kapa kit. All these methods are relative to some standard. Once you know what factor produces the desired clusters, the rest of the game is just consistency. We've started usuing fixed...
Forum: 454 Pyrosequencing 07-28-2012, 01:38 PM
Replies: 24
Views: 5,927
Posted By DNA_Dan
We are doing the same except using Lib-L kits on...

We are doing the same except using Lib-L kits on a FLX in LV format. Key is reducing that primer amount. Also I think if you are sequencing all the same amplicon, you have higher signal inteference...
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