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Forum: Illumina/Solexa 10-21-2016, 06:47 AM
Replies: 4
Views: 1,436
Posted By sweetph3
If you are more interested in the smaller size...

If you are more interested in the smaller size range of your library, keep in mind that the smaller fragments will cluster more efficiently than the larger fragments anyway, so you'll see more...
Forum: Sample Prep / Library Generation 04-08-2016, 06:30 AM
Replies: 14
Views: 12,282
Posted By sweetph3
Any time I've had issues, especially if the gDNA...

Any time I've had issues, especially if the gDNA was prepared by another group, I start off with the MoBio Power Clean now. It hasn't failed me yet. I think some samples have inhibitors that carry...
Forum: Illumina/Solexa 07-22-2015, 12:55 PM
Replies: 2
Views: 1,040
Posted By sweetph3
I know this is too late, but I had a similar...

I know this is too late, but I had a similar issue a while back and Tech Support told me it's not possible to change the run parameters once the MiSeq run has started.
Forum: Sample Prep / Library Generation 07-02-2015, 08:31 AM
Replies: 5
Views: 1,541
Posted By sweetph3
I thought Nextera index primers had some sort of...

I thought Nextera index primers had some sort of nucleotide modification. I didn't realize regular primers would work. If so, great! Thanks!
Forum: Sample Prep / Library Generation 07-01-2015, 10:49 AM
Replies: 5
Views: 1,541
Posted By sweetph3
16S V3-V4 Multiplexing >24 samples

I have a colleague who wants to amplify 16S V3-V4 and then have me multiplex and run on MiSeq. She at this point has 26 samples, but using Illumina's protocol for indexing with Nextera index...
Forum: Illumina/Solexa 06-12-2015, 11:37 AM
Replies: 30
Views: 8,620
Posted By sweetph3
What is the fragment size of the PhiX library? I...

What is the fragment size of the PhiX library? I haven't ever run it on a Bioanalyzer, so I don't know if it's relatively small or large. Since smaller fragments tend to cluster with better...
Forum: Sample Prep / Library Generation 01-12-2015, 11:34 AM
Replies: 7
Views: 5,766
Posted By sweetph3
Isolating bacterial mRNA from mixed prokaryotic + eukaryotic total RNA pool

My supervisor has already agreed to help a collaborator to sequence total RNA from a pool of plant and bacteria. They want me to do one library set for the plant mRNA (easy enough, capture poly-A...
Forum: Illumina/Solexa 11-25-2014, 06:59 AM
Replies: 7
Views: 2,241
Posted By sweetph3
I had run into issues with inhibitors in DNA...

I had run into issues with inhibitors in DNA preps when trying to make Nextera libraries. The only kit that removed the inhibitors was MoBio PowerClean DNA kit. Zymo and Qiagen didn't work. MoBio...
Forum: Sample Prep / Library Generation 07-11-2014, 01:33 PM
Replies: 2
Views: 1,408
Posted By sweetph3
Strange RNA Bioanalyzer trace, any thoughts?

We have only just started using the Bioanalyzer RNA 6000 Nano chips, and I haven't seen a good trace yet. Today's was very strange....

There is a main peak at ~40 seconds that blows the rest of...
Forum: Sample Prep / Library Generation 04-07-2014, 12:23 PM
Replies: 5
Views: 3,347
Posted By sweetph3
I also saw a larger than expected product. I ran...

I also saw a larger than expected product. I ran the Covaris to shear at 500 bp, and the final libraries had Bioanalyzer peaks at 780 bp. I don't have an explanation.
Forum: Illumina/Solexa 03-21-2014, 01:20 PM
Replies: 2
Views: 1,612
Posted By sweetph3
That's what I thought, thank you so much for your...

That's what I thought, thank you so much for your quick response!
Forum: Illumina/Solexa 03-21-2014, 12:58 PM
Replies: 2
Views: 1,612
Posted By sweetph3
silly question r.e. TruSeq stranded mRNA

Hi. I'll be receiving some library preps from a collaborator next week that were made with TruSeq Stranded mRNA kit. We will run them for her on our MiSeq.

She keeps emphasizing "STRANDED" in...
Forum: Illumina/Solexa 12-12-2013, 11:21 AM
Replies: 1
Views: 2,012
Posted By sweetph3
Can I run these as "Small RNA" on MiSeq?

I have a collaborator who amplified V3 regions using the following primers:
Forward

aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatctNNNNCCTACGGGAGGCAGCAG

Reverse (one of 10)
...
Forum: Sample Prep / Library Generation 10-16-2013, 01:24 PM
Replies: 2
Views: 3,743
Posted By sweetph3
MALBAC single cell WGA, any experience?

We have a difficult-to-grow sample of which we'll be doing genomic sequencing. We may need to do whole genome amplification of single cells. We've used Qiagen REPLI-g, but MDA has its...
Forum: Sample Prep / Library Generation 10-09-2013, 11:27 AM
Replies: 2
Views: 2,260
Posted By sweetph3
Using cDNA as input for TruSeq: Anyone tried this?

We have a cDNA sample that we would like to use to make a TruSeq library. We tried Nextera already, but Nextera due to how it tagments doesn't give us all of the 5' sequence. The cDNA is already...
Forum: Illumina/Solexa 09-23-2013, 06:22 AM
Replies: 44
Views: 19,793
Posted By sweetph3
We have been freezing 1 ml aliquots of 1N NaOH...

We have been freezing 1 ml aliquots of 1N NaOH (no mineral oil). We toss the aliquot after the 2nd thaw. Dilute to 0.2 N according to Illumina's protocol. Never see any problems with clustering...
Forum: Sample Prep / Library Generation 09-18-2013, 10:41 AM
Replies: 9
Views: 3,934
Posted By sweetph3
Yes, but we would like to see LARGER inserts. ...

Yes, but we would like to see LARGER inserts. We'd like to see the 2x300 cycles give more data than reading a 300bp (or smaller) insert 100% in either direction.

Any other ideas or advice?
Forum: Sample Prep / Library Generation 09-17-2013, 09:41 AM
Replies: 9
Views: 3,934
Posted By sweetph3
Size selection for Nextera libraries?

Hi. We have seen that for Nextera libraries, the smaller insert fragments cluster more efficiently than those with larger inserts. We ran our first 2x300 and most of the data is duplicated sequence...
Forum: Sample Prep / Library Generation 08-05-2013, 01:30 PM
Replies: 9
Views: 10,668
Posted By sweetph3
I had some sort of inhibitor in my genomic DNA. ...

I had some sort of inhibitor in my genomic DNA. I finally got some library after cleaning it up with MoBio Power Clean DNA Clean-Up Kit. My yield was 7.20 ng/ul. I'm also finally getting fragments...
Forum: Sample Prep / Library Generation 08-05-2013, 01:25 PM
Replies: 14
Views: 12,282
Posted By sweetph3
I finally had a chance to run 3 different...

I finally had a chance to run 3 different clean-up protocols and make Nextera with the resulting DNA. I used Qiagen plant DNeasy miniprep, MoBio Power Clean DNA Clean-Up Kit, and Zymo Research...
Forum: Sample Prep / Library Generation 07-08-2013, 01:20 PM
Replies: 14
Views: 12,282
Posted By sweetph3
Angry Failed tagmentation reaction for Nextera library?

Just had failures #2 and #3 using Illumina Nextera. :mad: I was able to run a sample from failure #1 of the post-zymo cleanup on Agilent 2100 Bioanalyzer this morning, and it looked almost identical...
Forum: Sample Prep / Library Generation 06-25-2013, 10:39 AM
Replies: 9
Views: 10,668
Posted By sweetph3
Question Nextera DNA library - low yield?

I just ran the Nextera DNA sample prep for the first time, following the protocol with the exception of using a Zymo column instead of the spin plate. Input was 50 ng genomic DNA (nuclear and...
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