PCR mix SYBR green

Date:________ Kit used:________ Oligo’s used:_________

Samples:_____________________________________________

Dilute the 2X RT (1ug RNA) reaction (40ul) 2.5X to 100 ul and use 10ul or 2.5 ul for Actin thus use 10% or 100ng RNA (cDNA).

Final concentrations: 1x buffer; 200uM d A,C,GTP; 400uM dUTP; 4mM MgCl2; 200nM each primer; 100ng DNA. Can use 2-6mM MgCl2 and 100-200nM oligo’s.

PCR Reaction:

Recommended ul per reaction: F.C.: ul used: Master Mix: PCR Cycles: Time\Temp:

10X PCR Buffer __5__ 1X _____ _____ One Denaturation:

25. mM MgCl2 __8 4mM _____ _____

10 mM dNTP’s __1___ 200nM _____ _____ _40__ step 1:

(2XUTP) step 2:

10uM 3’ oligo __1___ 200nM _____ _____

10uM 5’ oligo __1___ 200nM _____ _____

DNA (RT rxn) __10 200ng _____ _____

Sterile H2O up to 50ul _____ _____

Ampli Taq (5U/ul) __0.25__ 1.25U _____ _____

___________________________________

F.V.= 50. use _____ ul of master mix per sample.