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Forum: Sample Prep / Library Generation 06-15-2018, 07:42 AM
Replies: 5
Views: 1,008
Posted By ATϟGC
If agarose gel electrophoresis is your only...

If agarose gel electrophoresis is your only option for fragment size distribution, GelStar is also very sensitive and might be more suitable than Sybr Gold for using in the gel itself.

Gelstar use...
Forum: Sample Prep / Library Generation 06-04-2018, 11:34 AM
Replies: 3
Views: 612
Posted By ATϟGC
Hi again Sean, Sorry for making that...

Hi again Sean,

Sorry for making that guess/assumption. I forgot that eukaryotes have 16S RNA as well.

So it does appear that you have very low base diversity, you have rapidly declining q...
Forum: Sample Prep / Library Generation 06-01-2018, 04:56 AM
Replies: 3
Views: 612
Posted By ATϟGC
Hi Sean, Aside from PhiX, are you taking any...

Hi Sean,

Aside from PhiX, are you taking any measures to increase diversity in your amplicon libraries such as staggering/offsetting and/or adding other loci?


At a glance, your cluster...
Forum: General 05-31-2018, 04:28 AM
Replies: 6
Views: 1,105
Posted By ATϟGC
You are very welcome. Sounds like over-drying is...

You are very welcome. Sounds like over-drying is likely your main problem. If you are worried about ethanol contamination and want to minimize drying time you can remove as much ethanol as you can...
Forum: General 05-30-2018, 05:40 AM
Replies: 6
Views: 1,105
Posted By ATϟGC
I find it can be hard to get more than 80-90%...

I find it can be hard to get more than 80-90% recovery and I sometimes get as low as 50%.

Here are a few suggestions you can try for improving recovery:

-Do not dry the beads too long. If you...
Forum: Illumina/Solexa 09-06-2017, 06:12 AM
Replies: 7
Views: 1,015
Posted By ATϟGC
Is that judged from the thumbnail images or the...

Is that judged from the thumbnail images or the densities as measure in the flow cell chart of SAV?
Forum: Illumina/Solexa 09-06-2017, 05:19 AM
Replies: 7
Views: 1,015
Posted By ATϟGC
You might want to compare the clustering at the...

You might want to compare the clustering at the top (where sample was loaded) versus the bottom.

With typical cluster generation there are more clusters at the top than the bottom of the lanes...
Forum: Sample Prep / Library Generation 07-04-2017, 11:52 AM
Replies: 9
Views: 1,462
Posted By ATϟGC
Hi SNPsaurus, I think that they are just...

Hi SNPsaurus,

I think that they are just referring to the typical single-digest RAD method where restriction is followed by sonication or another shearing method to generate fragment size...
Forum: Sample Prep / Library Generation 07-04-2017, 05:13 AM
Replies: 3
Views: 830
Posted By ATϟGC
I have always thought of the NaCl (or another...

I have always thought of the NaCl (or another source of Na+ like Sodium acetate) as a co-factor required for an ethanol, isopropanol or PEG-base nucleic acid precipitation. NaCl might precipitate...
Forum: Sample Prep / Library Generation 06-29-2017, 04:37 AM
Replies: 9
Views: 1,462
Posted By ATϟGC
Here is an article that may be of interest to you...

Here is an article that may be of interest to you if you are looking to decrease costs and improve sequence quality:
...
Forum: General 05-18-2017, 12:02 PM
Replies: 2
Views: 1,118
Posted By ATϟGC
As you have mentioned, plant DNA barcoding to the...

As you have mentioned, plant DNA barcoding to the species level is rather imperfect to put it mildly. Incomplete lineage sorting and hybridization have led to the sharing of chloroplast haplotypes in...
Forum: Sample Prep / Library Generation 03-29-2017, 08:28 AM
Replies: 4
Views: 2,063
Posted By ATϟGC
The best method might depend largely on the taxon...

The best method might depend largely on the taxon or taxa you are using if there are abundant co-precipitating contaminants. I have attached a method I developed/modified for HMW extraction of DNA...
Forum: Sample Prep / Library Generation 02-17-2017, 06:28 AM
Replies: 8
Views: 1,716
Posted By ATϟGC
If RNA contamination is your issue the following...

If RNA contamination is your issue the following likely won't help you, but it might if you problem is contaminants:

For recalcitrant DNA extractions I highly recommend trying an CTAB...
Forum: Sample Prep / Library Generation 02-08-2017, 10:37 AM
Replies: 8
Views: 1,306
Posted By ATϟGC
Hi again, I usually see poor nanodrop-Qubit...

Hi again,

I usually see poor nanodrop-Qubit correlations when the A260/230 get below ~1.7. Here is a link to another thread discussing common contaminants and their effects on the spectra of...
Forum: Sample Prep / Library Generation 02-08-2017, 04:49 AM
Replies: 8
Views: 1,306
Posted By ATϟGC
What type of samples are you extracting from? I...

What type of samples are you extracting from? I ask this because your poor correlations could also be due to co-precipitants like polysaccharides that give you poor A260/230 values. Ideally, A260/230...
Forum: Complete Genomics 01-31-2017, 06:28 AM
Replies: 5
Views: 2,977
Posted By ATϟGC
I would expect that Sorbus species would have a...

I would expect that Sorbus species would have a fair amount of polysaccharides. I have had good success precipitating plant and animal DNA by CTAB dilution. Here are two articles with protocols:
...
Forum: Sample Prep / Library Generation 01-17-2017, 02:12 PM
Replies: 1
Views: 1,061
Posted By ATϟGC
Hi, The optimal loading concentration and...

Hi,

The optimal loading concentration and phiX % spike in will likely depend largely on the nucleotide sequence diversity at the start of your reads (on the HiSeq 2000 the first 12 bp is the most...
Forum: Sample Prep / Library Generation 08-19-2016, 08:19 AM
Replies: 14
Views: 6,492
Posted By ATϟGC
I'll echo ECO and melop in saying that I've tried...

I'll echo ECO and melop in saying that I've tried SPRI beads (Ampure and Seramag speedbeads) to clean up WGA products as well as genomic DNA from home-brew library prep and genomic DNA extraction...
Forum: Sample Prep / Library Generation 06-27-2016, 10:08 AM
Replies: 6
Views: 4,644
Posted By ATϟGC
Hello again, My experience with DNA...

Hello again,

My experience with DNA extraction from RNAlater preserved tissues was mostly plant-related but I am about to do some extractions from RNAlater preserved fish gills so I have been...
Forum: Sample Prep / Library Generation 06-15-2016, 08:01 AM
Replies: 6
Views: 4,644
Posted By ATϟGC
Hi again, If you have already tried soaking...

Hi again,

If you have already tried soaking the samples then I doubt that PBS would help you out.

If the problem is indeed guanidine-HCl or other chaotropic salts, I expect that those would be...
Forum: Sample Prep / Library Generation 06-14-2016, 05:03 AM
Replies: 6
Views: 4,644
Posted By ATϟGC
Howdy, Low A260/230 can be caused by many...

Howdy,

Low A260/230 can be caused by many contaminants including guanidine salts, phenolics, glycogen or other carbohydrates. In my experience if it is pervasive among different isolation...
Forum: Illumina/Solexa 05-18-2016, 10:41 AM
Replies: 23
Views: 3,988
Posted By ATϟGC
Reads coming from only half of your plates sounds...

Reads coming from only half of your plates sounds like there might have been a protocol-specific problem but ~3 million reads is pretty low so you may have had low-diversity and/or clustering...
Forum: Illumina/Solexa 03-18-2016, 06:11 AM
Replies: 10
Views: 1,779
Posted By ATϟGC
I apologize. You are right kmcarr. I think my...

I apologize. You are right kmcarr. I think my brain filled in a few of those grainy pixels and read density instead of intensity. That explains why the gradient scale is not a sensible number for...
Forum: Illumina/Solexa 03-17-2016, 06:54 AM
Replies: 10
Views: 1,779
Posted By ATϟGC
I think that the flow cell density chart may show...

I think that the flow cell density chart may show the signs of overclustering. I say this as your density increases from bottom to top. It should be highest at the bottom and decrease towards the...
Forum: Illumina/Solexa 02-25-2016, 06:58 AM
Replies: 27
Views: 2,837
Posted By ATϟGC
Yes the data did look normal in the sense that...

Yes the data did look normal in the sense that the ~5-7% of reads that passed filter were primarily (+99%) ddRAD loci. The sequence quality was poorer than we are used to, particularly in bases 6-12...
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