SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
Can you use PicoGreen in a Qubit assay? crader General 2 12-05-2012 10:35 AM
picogreen niceday General 0 11-04-2011 06:07 AM
Quality Control Library using QUBIT... help me! soleulloa Ion Torrent 1 09-01-2011 06:18 AM
PicoGreen Assay TUCF JSS Sample Prep / Library Generation 2 03-30-2011 02:19 PM
QuBit fluorimeter vs. Epoch? sehrrot Sample Prep / Library Generation 3 09-16-2010 11:09 AM

Reply
 
Thread Tools
Old 09-16-2010, 07:01 PM   #1
seqgirl123
Member
 
Location: U.S

Join Date: Oct 2008
Posts: 76
Default qubit/picogreen

I have a question about the qubit assay kit for measuring concentrations of dsDNA. The kit comes with 2 standards as tubes. The standards are labeled as 0ng/ul and 10ng/ul.

I calibrate the qubit with these standards just fine. First I make up the Picogreen mix, then take 10ul of each standard and dilute into 190ul of Picogreen mix, so the standards are diluted 1/20. After qubit is calibrated, I measure the standards in the kit, they read as <0.50ng/ml (for the 0ng/ul tube) and >600ng/ml (for 10ng/ul tube).

My question is how does the qubit calculate the 10ng/ul labeled standard tube as being greater than 600ng/ml when it is diluted 20-fold in the first place? Can someone show me the calculation of how it does this? I only want to know how it reads the standards as it does.
seqgirl123 is offline   Reply With Quote
Old 11-02-2013, 05:43 AM   #2
mmmm
Senior Member
 
Location: UK

Join Date: Jul 2013
Posts: 131
Default

when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?
mmmm is offline   Reply With Quote
Old 11-02-2013, 06:20 AM   #3
bstamps
Member
 
Location: University of Oklaoma

Join Date: Oct 2012
Posts: 40
Default

Well, from the Life Tech website...

The BR kit has a range of 100pg/uL to 1000ng/uL and the HS has a range of 10pg/uL to 100ng/uL. If you're on the extreme of the BR kit (Sample is 0.150 ish ng/uL), chances are the assay isn't going to read very accurately compared to the HS kit where you're further away from the lower bound. Same goes for HS if you're at the upper bound, which would place you comfortably in the middle of the BR kit.

We usually use the HS kit for PCR product and the BR kit for gDNA, as our yields for each usually sit in the middle of each kit's range.

Quote:
Originally Posted by mmmm View Post
when I used Qubit HS (high senstivity kit) for measuring gDNA conc., I got entirely different concentation compared to BR (broad range) kit. do you know, what is the difference between Qubit HS kit and Qubit BR kit for DNA conc.?
bstamps is offline   Reply With Quote
Old 11-02-2013, 06:26 AM   #4
mcnelson.phd
Senior Member
 
Location: Connecticut

Join Date: Jul 2011
Posts: 162
Default

The concentration values that are shown on the Qubit are the concentration of DNA in the tube that you are reading, and not of the original sample. This means that you need to take the value that it gives you in ng/ml and convert it to the ng/ul of your actual sample.

I don't remember the exact equation to do that off the top of my head, but it's given in the Qubit manual so I'd suggest you take a look at that. I can say that when using the HS DNA kit, if we use 2ul of sample, then the raw value that is given by the Qubit in ng/ml can be converted to ng/ul for the sample by just shifting the decimal point to the left (e.g. if the Qubit says the conc. is 50ng/ml, then the actual conc. of the sample is 5ng/ul).

This is the biggest issue we have with new people using the Qubit, they forget to convert the raw values to the actual conc. of the sample and that can have very bad downstream effects on library preparation.
mcnelson.phd is offline   Reply With Quote
Old 11-02-2013, 07:06 AM   #5
bstamps
Member
 
Location: University of Oklaoma

Join Date: Oct 2012
Posts: 40
Default

The instrument also lets you "calculate stock concentration" for each sample. Just click the "Calculate Stock Concentration" button after reading a sample, set the units to ng/uL (Or whatever else you want it to be), and set the amount in uL you put into the sample tube (Usually 2uL on our end). It will calculate the actual concentration of sample, after which you can click "save" and it will record the actual concentration onto the local memory.

You have to do this for each sample, which is a little annoying (Clicking save each time, it remembers the amount in uL and units desired), but it moves pretty quickly.
bstamps is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:52 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO