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Forum: Sample Prep / Library Generation 05-01-2013, 12:19 AM
Replies: 5
Views: 5,904
Posted By niceday
Out of interest what protocol are you using to...

Out of interest what protocol are you using to extract from ffpe? And do you use zylene?

Thanks.
Forum: Illumina/Solexa 08-09-2012, 07:08 AM
Replies: 1
Views: 1,761
Posted By niceday
I haven't been involved in next gen for almost a...

I haven't been involved in next gen for almost a year but I do remember ampure beads can go off. Have you tried a new fresh aliquot of your beads? Also you could try and ph you beads and then contact...
Forum: Core Facilities 06-25-2012, 06:59 AM
Replies: 16
Views: 8,999
Posted By niceday
LGC genomics Berlin

LGC genomics
Berlin
Forum: General 02-28-2012, 12:46 AM
Replies: 1
Views: 1,309
Posted By niceday
episonic shearing

I have heard due to the wavelength of the episonic that the shearing is non random.
This came from sales staff so I trust it as I would the devil himself.

Has anyone checked their sequence data...
Forum: Sample Prep / Library Generation 02-23-2012, 07:48 AM
Replies: 5
Views: 3,143
Posted By niceday
what levels are your yields? We run DNA...

what levels are your yields?
We run DNA extraction from blood, saliva and bucal swabs. This is for both genotyping and sequencing.
If you can give me some more info I can possibly advise.
...
Forum: Sample Prep / Library Generation 11-29-2011, 05:13 AM
Replies: 12
Views: 11,978
Posted By niceday
previously any samples sent to us that were...

previously any samples sent to us that were quantitated on a nanodrop would be requantitated on site. We would never take a nanodrop value as accurate.

If you have always used a nanodrop without...
Forum: Sample Prep / Library Generation 11-21-2011, 04:20 AM
Replies: 1
Views: 1,893
Posted By niceday
check out episonic. They have a different...

check out episonic.
They have a different machine for fragmenting DNA. It's a lot cheaper than covaris.
Forum: General 11-04-2011, 06:07 AM
Replies: 0
Views: 1,767
Posted By niceday
picogreen

has anyone tried using smaller volumes of working solution for picogreen? the protocol says make up to 200ul. I'm pretty sure the volume can be reduced but I would be interested in the cutoff below...
Forum: Sanger/Dye Terminator 10-25-2011, 10:17 AM
Replies: 16
Views: 6,702
Posted By niceday
Thanks Phillip. I've been doing nextgen for a few...

Thanks Phillip. I've been doing nextgen for a few years but have been asked to look at an established capillary setup to reduce costs.
My first call is to seqanswers before I start spending money...
Forum: Sanger/Dye Terminator 10-25-2011, 07:25 AM
Replies: 16
Views: 6,702
Posted By niceday
Hi Pmiguel, You mentioned you were doing...

Hi Pmiguel,

You mentioned you were doing 1/32nd reactions. I'm assuming you are either diluting down, reducing the reaction volume or both.
Are you able to give me any information on this?
I...
Forum: General 10-12-2011, 05:00 AM
Replies: 1
Views: 2,080
Posted By niceday
truseq exome enrichment

Sorry if this question has already been posted.

Has anyone tried to get around truseq's gel size selection when preparing libraries for exome enrichment?

We would normally use a 6 minute...
Forum: Sample Prep / Library Generation 10-04-2011, 01:12 AM
Replies: 9
Views: 5,052
Posted By niceday
do you have quantitation for pre and postPCR...

do you have quantitation for pre and postPCR enrichment?
DEpending on the number of cycles you should see an increase in the amount of library.
If the increase is low then the amount of properly...
Forum: Sample Prep / Library Generation 10-03-2011, 06:36 AM
Replies: 41
Views: 24,259
Posted By niceday
to ktabbada If you are seeing qPCR results...

to ktabbada

If you are seeing qPCR results that are 10% of your bioanalyser or Qubit readings and you successfully clustered using the qPCR concs then you should have your bioinformaticians scan...
Forum: Illumina/Solexa 09-08-2011, 06:39 AM
Replies: 7
Views: 1,920
Posted By niceday
I'm with pmiguel. Your library sound good. Even...

I'm with pmiguel. Your library sound good. Even if you are slightly below 10 nM you can alter your protocol slightly and get good clusters. Too many cycles gives too much redundancy.
Forum: SOLiD 09-08-2011, 12:39 AM
Replies: 1
Views: 1,246
Posted By niceday
You have too many beads. You should get about 1...

You have too many beads. You should get about 1 billion beads. 2 billion means you probably have too much library in your emulsion and your wfa will show high n2s and poor on axis.
It might be...
Forum: Epigenetics 09-05-2011, 05:51 AM
Replies: 8
Views: 5,495
Posted By niceday
It sounds like a ligation buffer problem. If the...

It sounds like a ligation buffer problem. If the ligation buffer isnt stored properly or has too many freeze thaw cycles it stops working. If your ligation efficiency is very low then the complexity...
Forum: Illumina/Solexa 09-02-2011, 01:56 AM
Replies: 10
Views: 3,971
Posted By niceday
I have seen gradients but not to this extent. ...

I have seen gradients but not to this extent.
It looks as if the gradient is from cluster formation because it wouldnt be so consistent for each cycle during sequencing.
Have you had your cbot...
Forum: Illumina/Solexa 08-15-2011, 03:43 AM
Replies: 4
Views: 9,312
Posted By niceday
overlapping PCR amplicons? Or you could speak...

overlapping PCR amplicons?
Or you could speak to agilent about producing RNA baits for the 50kb region and do the enrichment.
There will be other ways but these two are just off the top of my head.
Forum: General 07-27-2011, 05:34 AM
Replies: 3
Views: 1,956
Posted By niceday
Thanks for that. I'm trying to get a rough idea...

Thanks for that. I'm trying to get a rough idea of the number of sequence reads so I know if we have enough after the 25bp PF stage.
Forum: General 07-27-2011, 12:47 AM
Replies: 3
Views: 1,956
Posted By niceday
clusters per tile to clusters mm2

Hello,

Can someone tell me the conversion factor from cluster per tile to clusters per mm2. It would be very helpful.
Thanks.
Forum: Sample Prep / Library Generation 07-18-2011, 08:09 AM
Replies: 41
Views: 24,259
Posted By niceday
We do get qPCR results higher than Qubit and...

We do get qPCR results higher than Qubit and bioanalyser. I spoke to some-one at Kapa and they did say others had observed this. We use the qPCR as standard and we get reproducible results.
Forum: Sample Prep / Library Generation 07-12-2011, 11:57 PM
Replies: 2
Views: 2,145
Posted By niceday
What kit was used for chemical fragmentation? I...

What kit was used for chemical fragmentation?
I used a commercial kit for fragmentation. The time was 15 mins. I tried fragmenting for 10 mins and increased it by 1 min up to 15 mins. I got the same...
Forum: Sample Prep / Library Generation 07-12-2011, 01:08 AM
Replies: 49
Views: 69,833
Posted By niceday
if the beads dry to cracking it can be difficult...

if the beads dry to cracking it can be difficult to elute the DNA in the time mentioned in the protocol.

Some people make up 70% by eye and because 7ml of EtOH and 3ml of water has a volume less...
Forum: Illumina/Solexa 06-30-2011, 05:28 AM
Replies: 16
Views: 10,960
Posted By niceday
I would be more interested in the ligation...

I would be more interested in the ligation efficiency before PCR.
After PCR the amount of ligated material is much higher because PCR only amplifies up properly ligated library.

qPCR results...
Forum: Sample Prep / Library Generation 06-24-2011, 07:27 AM
Replies: 7
Views: 11,601
Posted By niceday
what does your peak look like before PCR and did...

what does your peak look like before PCR and did you cleanup using ampure? I have seen some extra peaks if my libraries are not completely clean of ampure beads. Usually at the higher end of the...
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