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Forum: 454 Pyrosequencing 09-05-2012, 09:56 AM
Replies: 13
Views: 3,072
Posted By Soulbee
I used to work with GS FLX about a year ago, and...

I used to work with GS FLX about a year ago, and I have had experienced many broken emulsions. We used blunt end tips because narrow end tips gave us drastically more broken wells. Also we had to...
Forum: 454 Pyrosequencing 03-25-2011, 07:36 AM
Replies: 4
Views: 2,012
Posted By Soulbee
Technically, it should work... I thought this...

Technically, it should work... I thought this through as well and I don't see why it wouldn't. When i called Roche, they weren't quite sure and have not yet gotten back to me about it...
Forum: 454 Pyrosequencing 03-02-2011, 04:57 AM
Replies: 4
Views: 2,911
Posted By Soulbee
It shouldn't matter which image you replace it...

It shouldn't matter which image you replace it with because as far as the software is concerned, whatever image you put in place of the first T flow will in fact be the T flow. The software knows...
Forum: 454 Pyrosequencing 03-01-2011, 07:02 AM
Replies: 6
Views: 4,105
Posted By Soulbee
I always thaw out my sequencing reagents at 4...

I always thaw out my sequencing reagents at 4 degrees overnight (from -20) and never have failed or saw any decrease in read length... could anything else be an issue?
Forum: 454 Pyrosequencing 03-01-2011, 06:22 AM
Replies: 7
Views: 1,725
Posted By Soulbee
Agreed. I had to also do many test runs before I...

Agreed. I had to also do many test runs before I got any replacement oils. Do you know if they are coming up with any new developments as far as the software or reagents go? I feel that compared to...
Forum: 454 Pyrosequencing 03-01-2011, 06:17 AM
Replies: 4
Views: 2,911
Posted By Soulbee
Why dont you try copying and pasting an image...

Why dont you try copying and pasting an image from another flow in the same run that has passed with a good brightness in place of the T image that has failed? With 454 GSFLX, the image files are...
Forum: 454 Pyrosequencing 03-01-2011, 05:59 AM
Replies: 27
Views: 10,940
Posted By Soulbee
hi, Ive had many cases with overenrichment with...

hi, Ive had many cases with overenrichment with FLX and most were due to broken emulsions, detectable by settled beads in the wells after ePCR. However i did have one or two samples with continuous...
Forum: 454 Pyrosequencing 02-28-2011, 10:47 AM
Replies: 9
Views: 2,796
Posted By Soulbee
After you did your second size selection, did you...

After you did your second size selection, did you confirm you size? I have had times where SV and LV oils do not correlate with each other as far as cpb goes.. which made the titration pointless....
Forum: 454 Pyrosequencing 02-28-2011, 07:34 AM
Replies: 4
Views: 2,103
Posted By Soulbee
it looks like not very much of your actual...

it looks like not very much of your actual library has amplified which makes me wonder if your adaptors/primers are annealed properly.. how was your enrichment?
Forum: 454 Pyrosequencing 02-09-2011, 07:51 AM
Replies: 11
Views: 3,591
Posted By Soulbee
We have also had much inconsistencies with our...

We have also had much inconsistencies with our emulsion oils between SV oil and MV/LV. With the MV, roche ended up replacing my reagents because they could not solve my problem of high enrichment %,...
Forum: 454 Pyrosequencing 02-09-2011, 07:37 AM
Replies: 9
Views: 2,796
Posted By Soulbee
did you perform a titration with SV oils? this...

did you perform a titration with SV oils? this could give you a better idea for cpb and you dont have to do a four point titration, just do two - one really low cpb and a higher one.
How long are...
Forum: 454 Pyrosequencing 02-09-2011, 07:27 AM
Replies: 7
Views: 3,143
Posted By Soulbee
I found that the longer you leave the melt...

I found that the longer you leave the melt solution in the tube to get the sequencing beads off, the more they will bind back to the enrichment beads. I do my first melt treatment, transfer the...
Forum: 454 Pyrosequencing 02-01-2011, 11:10 AM
Replies: 15
Views: 3,958
Posted By Soulbee
There is definitely a variability between the SV...

There is definitely a variability between the SV and LV kits. With the new LV oil, I see a distinct clear layer after the emulsion PCR, but with SV, there is not a clear layer in all of them. Not...
Forum: 454 Pyrosequencing 01-20-2011, 07:24 AM
Replies: 3
Views: 3,027
Posted By Soulbee
not sure what it is exactly, a stabilizer i...

not sure what it is exactly, a stabilizer i presume. anyway, tech support has told me that not spinning down the undissolved precipitate in the additive and adding the precipitate to our emulsion can...
Forum: 454 Pyrosequencing 12-16-2010, 05:33 AM
Replies: 15
Views: 3,958
Posted By Soulbee
I dont think so, because two of the four samples...

I dont think so, because two of the four samples were repeats, and have worked before in the past with LV emulsions.. We are going to try running the samples with LV oils instead. Thanks!
Forum: 454 Pyrosequencing 12-10-2010, 10:52 AM
Replies: 15
Views: 3,958
Posted By Soulbee
With the new LV oil, i have noticed a distinct...

With the new LV oil, i have noticed a distinct clear layer at the top, but I have only used a couple of these oils.

Broken emulsions can be distinguished from the non-broken ones by looking at...
Forum: 454 Pyrosequencing 12-10-2010, 06:24 AM
Replies: 15
Views: 3,958
Posted By Soulbee
I tested different lids (strip caps, adhesive and...

I tested different lids (strip caps, adhesive and two different kinds of heat seals) - The heat seals gave the best results and the adhesive one resulted in lots of broken wells. The new oil is...
Forum: 454 Pyrosequencing 12-01-2010, 04:27 AM
Replies: 15
Views: 3,958
Posted By Soulbee
Thanks for the input, we are going to also try...

Thanks for the input, we are going to also try using a different sealing method because the new oil formulations may be more sensitive to heat, which may be why im seeing lots of broken emulsions.
Forum: 454 Pyrosequencing 11-30-2010, 07:34 AM
Replies: 15
Views: 3,958
Posted By Soulbee
Hi, Im carrying out MV emPCR, (oil: 93777520;...

Hi, Im carrying out MV emPCR, (oil: 93777520; kit: 93786520) and a majority of my emulsion has come out broken. I have not had any problems in the past with broken emulsions and these are libraries...
Forum: 454 Pyrosequencing 11-16-2010, 07:06 AM
Replies: 2
Views: 2,558
Posted By Soulbee
We have not yet tried using gel extraction but...

We have not yet tried using gel extraction but had a similar idea of using E-gels prior to the Library immobilization step to size select then continue on with the protocol and immobilize the beads...
Forum: 454 Pyrosequencing 09-02-2010, 06:55 AM
Replies: 21
Views: 11,730
Posted By Soulbee
i'm using LV lot # 93799720 which has not given...

i'm using LV lot # 93799720 which has not given me any problems, and neither has the MV (but not sure what the Lot number is for the MV oil, sorry!)
Forum: 454 Pyrosequencing 08-10-2010, 10:21 AM
Replies: 21
Views: 11,730
Posted By Soulbee
Hey do you guys know if the sequencing kits are...

Hey do you guys know if the sequencing kits are in backorder right now and how long it may take to get them?

Also, during full processing of your sequencing data, its says "410 reads were capped"...
Forum: 454 Pyrosequencing 08-05-2010, 06:58 AM
Replies: 21
Views: 11,730
Posted By Soulbee
no, my problem is usually the opposite where too...

no, my problem is usually the opposite where too little beads are being washed off even though very small amount of template beads were added in the first place. If they are all being washed off and...
Forum: 454 Pyrosequencing 07-27-2010, 11:08 AM
Replies: 21
Views: 11,730
Posted By Soulbee
I ve been having a problem with emPCR as well...

I ve been having a problem with emPCR as well with the enrichment step. I used the same reagents and one LV oil from the same kits to process two different samples. During the enrichment step, there...
Forum: Sample Prep / Library Generation 07-27-2010, 10:35 AM
Replies: 6
Views: 2,766
Posted By Soulbee
I have also prepared cDNA libraries using rapid...

I have also prepared cDNA libraries using rapid library kit and protocol with MID adaptors. I actually elongated the chemical fragmentation step to 1 minute instead of 30 seconds because of the large...
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