SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 25 of 54
Search took 0.00 seconds.
Search: Posts Made By: ATϟGC
Forum: Sample Prep / Library Generation 04-01-2020, 05:47 AM
Replies: 11
Views: 1,083
Posted By ATϟGC
correction....inhibitors are typically only a...

correction....inhibitors are typically only a problem in round 1 of PCR. By the second round they are usually diluted enough that they do not cause problems.
Forum: Sample Prep / Library Generation 04-01-2020, 04:50 AM
Replies: 11
Views: 1,083
Posted By ATϟGC
I have used the SequalPrep Normalisation 96-well...

I have used the SequalPrep Normalisation 96-well plates for about 4-5 years now after the second and final round of PCR and they seem to work OK when you have strong and even amplification of your...
Forum: Bioinformatics 02-12-2020, 11:16 AM
Replies: 2
Views: 663
Posted By ATϟGC
The concatenate function of Seqkit...

The concatenate function of Seqkit [https://bioinf.shenwei.me/seqkit/usage/#concat] will concatenate sequences with matching ID's from two fasta files.

I converted your inputs into fasta format...
Forum: Metagenomics 11-26-2019, 11:14 AM
Replies: 2
Views: 2,205
Posted By ATϟGC
Have you tried or considered using a blocking...

Have you tried or considered using a blocking oligo (such as one with a C3 spacer) designed to reduce amplification of the "host" plant tissue? A quick look at an alignment of nucleotide data might...
Forum: Illumina/Solexa 11-26-2019, 11:07 AM
Replies: 3
Views: 1,002
Posted By ATϟGC
My guess is that you cannot use the "default"...

My guess is that you cannot use the "default" sequencing primers with your scheme.

You appear to have directly attached your gene-specific primer sequence directly to the i5 and i7 illumina...
Forum: Oxford Nanopore 10-23-2019, 08:28 AM
Replies: 2
Views: 2,580
Posted By ATϟGC
I have done a few large volume CTAB-Chloroform...

I have done a few large volume CTAB-Chloroform DNA extractions in polypropylene without any noticeable issues with the nucleic acids. I was told by someone with experience in a forensics lab that...
Forum: General 06-05-2019, 08:34 AM
Replies: 3
Views: 2,008
Posted By ATϟGC
I have tried the following protocol with insects...

I have tried the following protocol with insects and it works quite well in terms of purity and length:

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues
...
Forum: Sample Prep / Library Generation 05-24-2019, 08:32 AM
Replies: 6
Views: 1,273
Posted By ATϟGC
I tried to use limiting amount of SPRI beads...

I tried to use limiting amount of SPRI beads (Sera-Mag speedbeads and Commercial preparations diluted in their own buffer) for the normalization of amplicons prior to pooling without any success. I...
Forum: Illumina/Solexa 04-23-2019, 04:42 AM
Replies: 2
Views: 1,133
Posted By ATϟGC
A few questions that might help: What DNA...

A few questions that might help: What DNA polymerase are you currently using for PCR? Are your 16S primers tailed for high-throughput sequencing?

If inhibition is the problem, dilution will often...
Forum: Bioinformatics 04-02-2019, 05:07 AM
Replies: 7
Views: 892
Posted By ATϟGC
I will just echo cmbetts in saying that you...

I will just echo cmbetts in saying that you should always remove your oligo/primer sequences from Sanger and High-throughput sequencing data since they are artificial. You can sometimes find...
Forum: Illumina/Solexa 03-18-2019, 05:03 AM
Replies: 2
Views: 1,329
Posted By ATϟGC
Thanks Genomax. It is good to hear that it is...

Thanks Genomax. It is good to hear that it is comparable to other Illumina instruments and that dual indexes should mitigate the hopping.
Forum: Illumina/Solexa 03-13-2019, 06:24 AM
Replies: 2
Views: 1,329
Posted By ATϟGC
NovaSeq error rate?

I was wondering if anybody can quote the error rate for a NovaSeq? Even an idea of how it compares to MiSeq and HiSeq would be great as I can not seem to find any comparisons or hard numbers...
Forum: Sample Prep / Library Generation 11-22-2018, 10:11 AM
Replies: 4
Views: 1,118
Posted By ATϟGC
Since a lot of dsDNA fluorescent dyes have an...

Since a lot of dsDNA fluorescent dyes have an excitation maximum of about 490 nm (blue light) and and emission at about 520 nm (green light) You can make your own blue light epi-illuminator to excise...
Forum: Sample Prep / Library Generation 06-15-2018, 07:42 AM
Replies: 5
Views: 1,841
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: 1,059
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: 1,059
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: 2,080
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: 2,080
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: 2,172
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: 2,172
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: 2,215
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: 1,586
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: 2,215
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: 1,506
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: 3,883
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...
Showing results 1 to 25 of 54

 


All times are GMT -8. The time now is 01:49 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO