Preforming the PCR

Perform qPCR with 2X Hy-SYBR power mix (using a different kit requires protocol adjustments) on a 96-well plate

• Prepare a separate mix for each primer you are using. The SYBR mix is very viscous, so always prepare extra mix (~ x1.2) and mix well
• Primers for total yeast quantification: YEASTF (5-GAGTCGAGTTGTTTGGGAATGC-3); YEASTR (5-TCTCTTTCCAAAGTTCTTTTCATCTT T-3)[[[1]]]

For high specificity reaction

1. 2X Hy-SYBR power mix: 8ul
2. Forward primer (10uM): 0.4ul
3. Reverse primer (10uM): 0.4ul

• Prepare a separate tube with your diluted DNA using sterile DDW to achieve a concentration between 10-100ng in 11ul per well (totaling 20ul per reaction). All samples should be diluted to the same concentration. Always prepare a mix for all samples and extra.
• Prepare your plate by loading the SYBR-primer mixes first into all wells. Then, load the DNA.

QPCR thermal cycling conditions

File:PCR protocol.pdf