Hello,
I have a conventional primer pair and conventional mastermix.
I want to use this as a real-time assay on my CFX96 Real-time machine.
Can I add SYBR green or any other dye separately in the above and get real-time chemistry?
If yes, then any suggestions about the concentration of the dye that needs to be added for 25 and 50uL PCR reactions?
Any other ideas to convert conventional chemistry in to real-time chemistry?
Thanks in advance.
Farhan
How big is your product? RT-PCR usually amplifies small products ~150 bp.
The product is around 200. But how much dye should be added to make a real time PCR assay. Thanks
You should buy SYBR Green Master mix that has Taq dNTP and Sybr premixed. From conventional optimized PCR only use primers and set assay on real time PCR. You need to use thermal cycling program as suggested in SYBR Green master mix product insert by only varying the annealing temperature (Use Ta that you optimized in conventional PCR)
Dear Farhan, in the attachment you can find Bio-Rad SYBRGreen master mix protocol. The amounts and the program for CFX96 were shown in the document.
Best regards.
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