2 replies to this topic

[Newbie_10]

Quick and simple question.

I'm doing SybrGReen based qPCR for the first times and been optimizing my sample preparation. So sorry for my basic questions, bare with me!

I started with normal reaction volumes: 15 ul of mastermix  containing 12.5ul SybrGreen (+primers+water), and 10 ul of sample.
Total volume being 25ul/well.
Due to several reasons I have now increased the total reaction volume, the sample volume being 40ul.
Does this mean I have to use 60 ul of mastermix and increase my primer concentration accordingly although the amount of template remains the same?

All answers are appreciated. Thx!

[tea-test]

Hi,

yes in order to have everything like before you have to increase the reaction volume to a final of 100µl and I would leave the primer concentration (not amount!) as before, independent from the amount of template being used. so this would mean 50µl MM + 10µl primer + 40µl template.

Are you using the 4 fold amount of template or do you just dilute the template 4 fold?

And be aware that not all real time cyclers can handle such big reaction volumes (like in ABI instruments equipped with a fast block the maximum rxn volume is 30µl)!

[josurb]

Newbie_10, on Mar 26 2010, 05:53 AM, said:

Quick and simple question.

I'm doing SybrGReen based qPCR for the first times and been optimizing my sample preparation. So sorry for my basic questions, bare with me!

I started with normal reaction volumes: 15 ul of mastermix  containing 12.5ul SybrGreen (+primers+water), and 10 ul of sample.
Total volume being 25ul/well.
Due to several reasons I have now increased the total reaction volume, the sample volume being 40ul.
Does this mean I have to use 60 ul of mastermix and increase my primer concentration accordingly although the amount of template remains the same?

All answers are appreciated. Thx!

Why did you go away from the 25ul volume reaction?  When I set up a sample, I set it up in Triplicate reactions.  I use 34.65ul of DNA Sample, 41.25 SyberGreen, 3.3ul FOR Primer, 3.3ul REV Primer (82.5ul total) and then aliquot 25ul into three separate wells on the plate.  There's extra simply for pipetting error.  The important thing to remember I guess if you continue on the route your on is that SyberGreen comes 2x, and should be diluted no more/no less to 1x, so be sure it's at 1x.   Also, as mentioned by the other poster here, make sure your primer concentrations remain the same.  If you change the total volume of the reaction but not the primer volume or concentration, then you don't have the same type of reaction anymore.   Hope this helps.