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Forum: Sample Prep / Library Generation 09-28-2019, 12:46 PM
Replies: 2
Views: 698
Posted By Carcharodon
Jumping in to address my own question: The...

Jumping in to address my own question:

The fact that this pattern appears consistently indicates some systematic error/issue. Looking back at old gels, I noticed that some of my digests showed...
Forum: Sample Prep / Library Generation 09-27-2019, 02:52 PM
Replies: 2
Views: 698
Posted By Carcharodon
Is this library overamplification? Is this problematic?

Aloha all,

I definitely DO have library overamplification in some of my libraries (as evidenced by a secondary peak, likely the result of the "daisy-chaining" effect). But that's not really what...
Forum: Sample Prep / Library Generation 09-10-2019, 11:22 PM
Replies: 5
Views: 982
Posted By Carcharodon
Interesting. I'll look into this a bit more,...

Interesting. I'll look into this a bit more, thanks.
Forum: Sample Prep / Library Generation 09-09-2019, 04:55 PM
Replies: 5
Views: 982
Posted By Carcharodon
Thanks for the response, luc! I'm currently...

Thanks for the response, luc! I'm currently working through a ddRAD protocol (my second library). Long story short I have digested DNA w/ ligated adapters. I bead-cleaned them after pooling, but...
Forum: Sample Prep / Library Generation 09-08-2019, 11:16 PM
Replies: 5
Views: 982
Posted By Carcharodon
Why is it commonly recommended to Speed-Vac without heat?

Especially when it comes to Illumina libraries, why is it commonly recommended to Speed-Vac without heat? Is this to prevent (or slow down) potential DNase activity?
Forum: Sample Prep / Library Generation 08-21-2019, 04:37 PM
Replies: 2
Views: 846
Posted By Carcharodon
Thanks, that's reassuring.

Thanks, that's reassuring.
Forum: Sample Prep / Library Generation 08-18-2019, 10:42 PM
Replies: 6
Views: 2,321
Posted By Carcharodon
I use the Biotium AccuClear system, and for that...

I use the Biotium AccuClear system, and for that you simply mix the DNA with the dye/buffer mixture by pipetting and allow it to sit for ~ 5 mins. I would bet good money that the same approach would...
Forum: Sample Prep / Library Generation 08-18-2019, 10:30 PM
Replies: 2
Views: 846
Posted By Carcharodon
Removal of carry-over EtOH after bead purification of DNA

I recently purified some DNA (double-digest of genomic DNA) with SPRI beads, and I don't believe I let them dry long enough to get rid of excess EtOH used during the rinse step before eluting the DNA...
Forum: Bioinformatics 08-28-2017, 06:12 PM
Replies: 8
Views: 1,320
Posted By Carcharodon
What do you guys think about AMD's Threadripper...

What do you guys think about AMD's Threadripper 1950X for these kinds of builds? $1,000 for the processor with 16 cores/32 threads, can be overclocked stable at 4.0 GHz? Good value?

Not sure how...
Forum: Sample Prep / Library Generation 08-02-2017, 07:21 PM
Replies: 2
Views: 2,022
Posted By Carcharodon
1) I've done ddRAD, and a couple of other folks I...

1) I've done ddRAD, and a couple of other folks I know have also done ddRAD, and we just use standard desalting. The HPLC purification that is often recommended is considerably more expensive, and...
Forum: Bioinformatics 07-13-2017, 06:09 PM
Replies: 0
Views: 884
Posted By Carcharodon
STACKS and PE Illumina Data (ddRAD)

I have a dataset from a ddRAD library (Illumina HiSeq4K, 150PE). I'm playing around with STACKS, and noticed that it has some limitations when dealing with paired-end data from ddRAD datasets.
...
Forum: Bioinformatics 07-12-2017, 12:09 PM
Replies: 0
Views: 840
Posted By Carcharodon
ddRAD, STACKS, and Trimming

Please disregard/delete. :)
Forum: Sample Prep / Library Generation 07-12-2017, 11:48 AM
Replies: 19
Views: 24,709
Posted By Carcharodon
This is probably better left for someone else to...

This is probably better left for someone else to answer. I don't have any real experience with this protocol. I've gone toe-to-toe with the beads more than a few times, but nested PCR is another...
Forum: Sample Prep / Library Generation 07-11-2017, 03:12 PM
Replies: 19
Views: 24,709
Posted By Carcharodon
I don't think it should be much of a problem,...

I don't think it should be much of a problem, unless I'm misunderstanding something. If the lower ratio is a concern, I would recommend taking one (or a few) of your PCR products and trying both...
Forum: Sample Prep / Library Generation 07-11-2017, 02:23 PM
Replies: 19
Views: 24,709
Posted By Carcharodon
I see! Well, I'd recommend using a ratio slightly...

I see! Well, I'd recommend using a ratio slightly less than 1.8x. I'd lower that to 1.5x or so and see if it makes a difference (30 uL AmpureXP to 20 uL ladder dilution).
Forum: Sample Prep / Library Generation 07-11-2017, 01:14 PM
Replies: 6
Views: 2,651
Posted By Carcharodon
I actually hadn't noticed, but the fin-clips I...

I actually hadn't noticed, but the fin-clips I collected were of different sizes (up to the size of a thumb-nail for adults, and maybe as small as half the area of a pinky-nail for neonates.

I...
Forum: Sample Prep / Library Generation 07-11-2017, 01:07 PM
Replies: 19
Views: 24,709
Posted By Carcharodon
We would need to know what the conditions of your...

We would need to know what the conditions of your test were and which lanes correspond to which treatment before we could really answer your question. Could you tell us these things?

Also, from...
Forum: Sample Prep / Library Generation 07-10-2017, 02:15 PM
Replies: 6
Views: 2,651
Posted By Carcharodon
There's a lot to unpack here, but I can give some...

There's a lot to unpack here, but I can give some advice!

Freezing immediately is great, so long as you have access to those temperatures at the time of sampling. If you can take along, say, some...
Forum: Sample Prep / Library Generation 07-04-2017, 03:51 PM
Replies: 3
Views: 1,389
Posted By Carcharodon
I think that the sodium ions, in this case, may...

I think that the sodium ions, in this case, may be somehow facilitating the binding of DNA to the carboxylated surface of the beads rather than playing a large role in the actual precipitation.
Forum: Sample Prep / Library Generation 07-02-2017, 09:56 PM
Replies: 3
Views: 1,389
Posted By Carcharodon
I might be stating the obvious here, but why not...

I might be stating the obvious here, but why not try 2.5M NaCl with 8% PEG? I can't explain why the other protocols seem to be working, but I really question whether it's necessary to reduce NaCl...
Forum: Sample Prep / Library Generation 06-28-2017, 05:26 PM
Replies: 9
Views: 2,051
Posted By Carcharodon
Hey tugecko, I'm doing ddRAD as well....

Hey tugecko,

I'm doing ddRAD as well. Size-selection with beads tends to leave you with a relatively wide range of fragment sizes (folks in my lab do ezRAD quite a bit, and this is the method they...
Forum: Sample Prep / Library Generation 06-01-2017, 08:21 PM
Replies: 3
Views: 2,344
Posted By Carcharodon
A little bit of EtOH carry-over is preferable to...

A little bit of EtOH carry-over is preferable to loss, which can happen if you over-dry your beads. And that is a very, very fine line.

I would actually reduce your dry times to below 5 minutes...
Forum: Sample Prep / Library Generation 05-28-2017, 10:00 PM
Replies: 10
Views: 5,957
Posted By Carcharodon
I find that over-drying the beads is incredibly...

I find that over-drying the beads is incredibly easy to do, and will cause massive reductions in yield. For smaller volumes of bead, I don't recommend drying for more than two minutes.

Reducing...
Forum: Sample Prep / Library Generation 05-15-2017, 04:30 PM
Replies: 8
Views: 2,365
Posted By Carcharodon
Hi ymilesz, Sorry for the late response. At...

Hi ymilesz,

Sorry for the late response. At first I actually ran 50uL reactions (due to low DNA concentrations in the elute), and then I tried running 25uL reactions.

Interestingly, the ones...
Forum: Sample Prep / Library Generation 04-23-2017, 09:19 PM
Replies: 8
Views: 2,365
Posted By Carcharodon
Well, I originally followed the protocol laid out...

Well, I originally followed the protocol laid out by Peterson et al. (2012), where digested DNA with ligated adapters were size-selected (w/ Pippin-Prep) and then went through PCR.

3 or 4 out of...
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