Editing 2X qPCR Mix Protocol (section)

=== '''Taqman Mix''' ===

You can also make a similar mix using a taqman probe instead of SYBR green. While the probes are expensive (over $100 each), they can lead to more reliable results, and eliminate the need for normalization based on the amplicon length when doing comparisons.

AmpErase® uracil-N-glycosylase (UNG) is a 26-kDa recombinant enzyme encoded by the Escherichia coli uracil-N-glycosylase gene. This gene has been inserted into an E. coli host to direct expression of the native form of the enzyme (Kwok and Higuchi, 1989). UNG acts on single- and double-stranded dU-containing DNA. It acts by hydrolyzing uracil-glycosidic bonds at dU-containing DNA sites. The enzyme causes the release of uracil, thereby creating an alkali-sensitive apyrimidic site in the DNA. The enzyme has no activity on RNA or dT-containing DNA (Longo et al., 1990).

TaqMan Assays For TaqMan® assays, AmpErase® UNG treatment can prevent the reamplification of carryover PCR products from previous PCR reactions. When dUTP replaces dTTP in PCR amplification, AmpErase UNG treatment can remove up to 200,000 copies of amplicon per 50-μL reaction.

Note: AmpErase UNG (also abbreviated as UDG for uracil-DNA-glycosylase) is included in some Applied Biosystems TaqMan master mix formulations and can also be purchased individually. When purchasing TaqMan master mixes, check the product information to see if the master mix contains AmpErase UNG.

All TaqMan Gene Expression Assays require: • Three components:

– 1 to 100 ng of cDNA sample (converted from RNA) per well, with all wells in a study having the same amount of cDNA.

– 20✕ Gene Expression Assay Mix (specific for each target). Each assay mix consists of two unlabeled PCR primers and a FAM™ dye-labeled TaqMan® MGB (minor groove binder) probe in a preformulated 20✕ mix. 1✕ final concentrations are '''250 nM''' for the probe and '''900 nM '''for each primer.

– TaqMan® Gene Expression Master Mix, TaqMan® Universal PCR Master Mix (with or without AmpErase® UNG), or TaqMan® Fast Universal PCR Master Mix, No AmpErase® UNG.