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Forum: Sample Prep / Library Generation 04-28-2011, 05:51 AM
Replies: 3
Views: 2,689
Posted By LMcSeq
Agreed-PicoGreen is intended to measure double...

Agreed-PicoGreen is intended to measure double stranded DNA. If the shearing resulted in any single stranded material, that would not be detected.
Forum: Sample Prep / Library Generation 01-18-2011, 01:26 PM
Replies: 5
Views: 3,520
Posted By LMcSeq
We've used the Pippin prep for size selection...

We've used the Pippin prep for size selection then cleaned up with AMPure at the end and had great success. Maybe you can fine tune your size selection step?
Forum: Sample Prep / Library Generation 11-05-2010, 11:04 AM
Replies: 46
Views: 24,019
Posted By LMcSeq
Do you purify the product after it comes out of...

Do you purify the product after it comes out of the Pippen Prep? There's ethidium bromide in there...perhaps you just need to run it through a column or add more volume of sample to your PCR.
Forum: 454 Pyrosequencing 11-05-2010, 11:03 AM
Replies: 3
Views: 1,816
Posted By LMcSeq
If there are 0 wells for control beads, then...

If there are 0 wells for control beads, then there is likely one of 2 problems:
1. the control beads weren't added (likely)
2. there is a problem with the instrument (only if you didn't get...
Forum: 454 Pyrosequencing 11-01-2010, 05:30 AM
Replies: 7
Views: 3,481
Posted By LMcSeq
I've heard that the white you're seeing isn't...

I've heard that the white you're seeing isn't beads--it can be a precipitate from the NaOH that is added. Don't kill yourself to get this off, especially if you're collecting enough beads.
However,...
Forum: 454 Pyrosequencing 10-18-2010, 10:34 AM
Replies: 28
Views: 11,541
Posted By LMcSeq
Does anyone have experiences to share about the...

Does anyone have experiences to share about the REMe on the Hamilton? Just got our Hamilton installed and we're intending to get the deck set up soon for the REMe.
Forum: 454 Pyrosequencing 09-17-2010, 03:30 PM
Replies: 5
Views: 2,270
Posted By LMcSeq
Sequencing kits were on backorder for a few...

Sequencing kits were on backorder for a few weeks, but they've now been released. Haven't had any recent trouble getting emPCR kits in the states.
Forum: Sample Prep / Library Generation 07-27-2010, 01:49 PM
Replies: 6
Views: 3,097
Posted By LMcSeq
We've seen longer libraries in general with the...

We've seen longer libraries in general with the rapid kit. I don't think it would be any different between the regular and cDNA kits.
Forum: Sample Prep / Library Generation 07-27-2010, 08:25 AM
Replies: 6
Views: 3,097
Posted By LMcSeq
There's been rumor that the adapter in the rapid...

There's been rumor that the adapter in the rapid library kit makes the libraries look much longer because of the FAM label. Try running the adapter alone on the Bioanalyzer and see what it looks...
Forum: 454 Pyrosequencing 06-14-2010, 05:46 AM
Replies: 28
Views: 11,541
Posted By LMcSeq
I've heard that Beckman is working on it and will...

I've heard that Beckman is working on it and will be supporting the REMe going forward.
Forum: 454 Pyrosequencing 05-14-2010, 01:06 PM
Replies: 13
Views: 3,335
Posted By LMcSeq
We've had the same problem.

We've had the same problem.
Forum: 454 Pyrosequencing 05-12-2010, 01:45 PM
Replies: 2
Views: 1,747
Posted By LMcSeq
8kb is more than enough

8kb is more than enough
Forum: 454 Pyrosequencing 05-10-2010, 01:22 PM
Replies: 13
Views: 3,335
Posted By LMcSeq
At the user's group meeting they said that there...

At the user's group meeting they said that there is a large backorder on sequencing reagents. They have tightened up their quality requirements from vendors.
Forum: General 04-29-2010, 03:15 PM
Replies: 2
Views: 2,349
Posted By LMcSeq
Which size range in best for the new rapid...

Which size range in best for the new rapid library prep?
Forum: 454 Pyrosequencing 04-22-2010, 11:25 AM
Replies: 42
Views: 13,889
Posted By LMcSeq
Did you use the Lib-L kit or Lib-A kit? Lib-A is...

Did you use the Lib-L kit or Lib-A kit? Lib-A is for amplicons.
Forum: Illumina/Solexa 04-17-2010, 10:12 AM
Replies: 4
Views: 3,911
Posted By LMcSeq
Try cleaning the bayonette cartridge in the...

Try cleaning the bayonette cartridge in the bioanalyzer and rerunning first. We sometimes get shifts in samples and it's just because it needs a good cleaning. Soak it in molecular grade water...
Forum: General 03-31-2010, 11:03 AM
Replies: 0
Views: 2,310
Posted By LMcSeq
Next Gen ISO 17025 Labs?

Hi all,
We're working on trying to get our next gen sequencing lab accredited to ISO 17025 (I know, we're crazy :eek:). We have two 454 instruments and intend to get a HiSeq this year.

Is anyone...
Forum: Sample Prep / Library Generation 03-30-2010, 07:44 PM
Replies: 4
Views: 3,227
Posted By LMcSeq
What about running another gel and selecting only...

What about running another gel and selecting only the size you want?
Forum: 454 Pyrosequencing 03-18-2010, 11:16 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
Phillip-they're all the same to me. People who...

Phillip-they're all the same to me. People who don't have answers to questions that they should know. :)

The capture bead is the white bead.
Forum: 454 Pyrosequencing 03-18-2010, 06:53 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
I spoke with my FAS who explained that the...

I spoke with my FAS who explained that the capture beads are better at holding onto the library frags.
We've had some runs that have also had higher mixed/dot and short reads. It started before the...
Forum: 454 Pyrosequencing 03-17-2010, 06:02 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
SeqMonster: Keep in mind that the DNA capture...

SeqMonster: Keep in mind that the DNA capture beads are now much more efficient than they previously were. You may need to adjust your cpb input if you aren't doing titration to optimize. We used to...
Forum: 454 Pyrosequencing 03-16-2010, 06:52 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
Cambridge454--I get long frag lengths with using...

Cambridge454--I get long frag lengths with using the Covaris as well. If you adjust the nebulization, I would add more time rather than change the pressure. Try doing a time course study (that's what...
Forum: 454 Pyrosequencing 03-09-2010, 06:39 AM
Replies: 3
Views: 1,922
Posted By LMcSeq
I think they recently internally recalled some...

I think they recently internally recalled some emPCR kits, so those were on backorder for a bit.
Forum: 454 Pyrosequencing 03-04-2010, 09:03 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
That's what I suspect is the problem, but I'm not...

That's what I suspect is the problem, but I'm not sure how to effectively troubleshoot it with the new rapid protocol since the chip isn't run until the end of all of the steps are complete. I don't...
Forum: 454 Pyrosequencing 03-04-2010, 08:09 AM
Replies: 69
Views: 25,912
Posted By LMcSeq
Hi Phillip, Thanks for your reply. We've had...

Hi Phillip,
Thanks for your reply. We've had our 454 FAS out and he said that its likely a problem in library prep or emPCR based on the output from the support tool (which customers can't view...
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