Self-made qRT-PCR enzyme-/mastermix? - (Feb/17/2006 )

Does anybody know how to mix an enzyme-/mastermix for RTQ-PCR by yourself? I will use the RTQ-PCR a lot and so my boss decided to buy a good heat-activated polymerase and the sybr-green and prepare the mastermix by ourselves as that would be cheaper than buying kits as we did before. So I have to find out how to do it and would be very grateful for a good protocol.

Please don't tell me to use kits. I know that they are easy to use and not that much more expensive. But unfortunately I am not in charge of my groups budget and my boss doesn't want to use kits.

Thanks for your help!

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This is the reaction mix I always use.

Master Mix for 1 (one) Real-Time PCR reaction (25ul total volume):

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H2O = 13,625ul (Nuclease free PCR-grade water)
10xBuffer = 2,5ul (1x Final concentration)
10nM dNTP mix = 0,5ul (0,8mM Final concentration - [G, A, T, C] )
50% Glycerol = 4ul (8% Final concentration)
ROX = 0,375ul (30nM Final concentration - Reference dye)
SYBR Green = 0,75ul (x0,5 Final concentration)
Taq DNA Polymerase = 0,25ul (2,5U per reaction)
Primer = 1ul (50-150nM Final concentration)
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Add 2ul of DNA template

*Glycerol diluted with H20
*SYBR Green diluted with DMSO 1:2000 before use
=> You can make a x100 dilution stock, which you can defrost and dilute x20 before each reaction.
*ROX diluted with H2O 1:500 before use

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There was a huge discussion on this sort of thing a few weeks ago. Check it out here:

http://www.protocol-online.org/forums/inde...topic=13200&hl=

-Hank

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So how much are you paying for the commercial MM now?

I routinely get std dev +/- 0.1 ct with commercial mixes.

-Matt

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Hey Matt, what commercial mix do you go with?

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I go with what's cheapest for me, which is Invitrogen SYBR w/ROX and UNG. I get a special deal that allows me to purchase 12.5 ml 2XMM for $300.

-Matt

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Because I'am very curious ......what deviations you get from you homemade mixtures...????
...and....are u using HotStart Taq for that application????

Greetz

K_R

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From what I have read (from that biotechniques SYBR paper that is posted in the other thread and on this board), you can get as much as ~0.5 ct variation, which is unsatisfactory for accurate quantification.

-Matt

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Thas also my experience.... I started a thread some weeks ago about this topic....so.....
1) You can use EVERY Taq with the supplied buffer by just adding sybrgreen in a 1:20.000 dilution
2) I tried 3 different taqs from Ferments....the one with BSA gave the best results...

BUT

you get Cts +/- around 0.3 or higher......so you need triplicates
....and thats the point....for a normal regulation in vivo....maybe around 50%.....it is not sufficient

I did siRNA-Experiments and got downkocks of around 80-90%....for that, the Homemade-Mixes where absolutly sufficiant.....

There are some Companies that sell there HotStart-MM in kombination with SybrGreen .....
They are all HotStart-Mixes.....so when you calculate the price you will see: I doesn't make sense to make your own mix...the components are more expensive than the mix..... I hope we will get good results with that "semi-commercial" solution


Greetz

K_R

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