Quantify Library before Cluster Generation

kwebb · 05-13-2009, 08:21 AM

Hello,

I'm working with genomic library prep kit for single ended reads. How do people quantify your library prior to cluster generation? The illumina protocol says to measure the absorbance at 260nm. However, I get very similiar 260nm readings but very different intensities when I run the libraries on an agarose gel. Does anyone have any suggestions?

Thank you,
Kristen

huguesparri · 05-14-2009, 02:10 AM

Hello,

We use Agilent DNA1000 chip to measure ou library concentration and size.
Usually, it's acurate enough for cluster number standardization.

H.

BioHak · 05-15-2009, 07:26 AM

We've been using the DNA1000 chips to quant our samples, has anyone here used the new High-sensitivity chips available from Agilent?

Laurence Game · 06-03-2009, 03:02 AM

The Bioanalyser high sensitivity DNA kit is not yet released in the UK. It will be in the next week or 2.

We quanitfy libraries using Picogreen and the Qubit. It takes 10min and is very reliable.

Laurence

huguesparri · 06-03-2009, 03:52 AM

And how do you get the size of your library using Qubit?

H.

Laurence Game · 06-03-2009, 03:57 AM

The question was about QUANTIFICATION. We quantify using Qubit.
We run a DNA chip on the Bioanalyser to check the profile and possible contaminants.

Is that clearer?

huguesparri · 06-03-2009, 04:07 AM

Yes, sure.
Sorry for being dumb

H.

captainentropy · 06-22-2009, 06:01 PM

After the PCR I ususally have enough DNA to spec by Nanodrop. But I also use a Qubit. The Bioanalyzer isn't very good for determining concentration (or so the facility running it says).

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05-13-2009, 08:21 AM	#1
kwebb Member Location: Wahington, DC Join Date: Jul 2008 Posts: 21	Quantify Library before Cluster Generation Hello, I'm working with genomic library prep kit for single ended reads. How do people quantify your library prior to cluster generation? The illumina protocol says to measure the absorbance at 260nm. However, I get very similiar 260nm readings but very different intensities when I run the libraries on an agarose gel. Does anyone have any suggestions? Thank you, Kristen

06-03-2009, 03:02 AM	#4
Laurence Game Junior Member Location: London, UK Join Date: May 2008 Posts: 5	Quantify Library before Cluster Generation The Bioanalyser high sensitivity DNA kit is not yet released in the UK. It will be in the next week or 2. We quanitfy libraries using Picogreen and the Qubit. It takes 10min and is very reliable. Laurence

05-14-2009, 02:10 AM	#2
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Hello, We use Agilent DNA1000 chip to measure ou library concentration and size. Usually, it's acurate enough for cluster number standardization. H.

05-15-2009, 07:26 AM	#3
BioHak Member Location: PharmLand -- Philadelphia Join Date: Aug 2008 Posts: 13	We've been using the DNA1000 chips to quant our samples, has anyone here used the new High-sensitivity chips available from Agilent?

06-03-2009, 03:52 AM	#5
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	And how do you get the size of your library using Qubit? H.

06-03-2009, 03:57 AM	#6
Laurence Game Junior Member Location: London, UK Join Date: May 2008 Posts: 5	The question was about QUANTIFICATION. We quantify using Qubit. We run a DNA chip on the Bioanalyser to check the profile and possible contaminants. Is that clearer?

06-03-2009, 04:07 AM	#7
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Yes, sure. Sorry for being dumb H.

06-22-2009, 06:01 PM	#8
captainentropy Member Location: San Francisco Bay Area Join Date: Mar 2009 Posts: 89	After the PCR I ususally have enough DNA to spec by Nanodrop. But I also use a Qubit. The Bioanalyzer isn't very good for determining concentration (or so the facility running it says).