RT-qPCR

Reverse Transcription

 * Add:
 * 1ul random hexamer primer
 * 11ul RNA/dH2O (1ug total RNA)
 * 1ul dNTPs (10mM each)


 * Heat mixture to 65°C for 5min and chill on ice.


 * Add:
 * 4ul 5X first-strand buffer
 * 2ul 0.1M DTT


 * Incubate @ 25°C for 2 min


 * Add: 1ul superscript II RT and incubate as follows: 25°C for 10 min, 42°C for 50 min, 70°C for 15 min.

qPCR

 * Dilute cDNA (ex 1:10, 1:100, 1:1000)
 * Must optimize this for each gene, 1:100 works well for Fba1

95°C 15min
 * Add 3ul cDNA per well.
 * Add 5ul 2X SYBRgreen mix and 2ul primer mix (70mM final concentration of each primer). **Make this as a master mix for each primer set you are looking at.
 * After sealing plate, spin down 30 sec at 500rpm in Welch lab centrifurge.
 * qPCR as follows:

95°C 15sec

60°C 30sec (40 cycles of steps 2 and 3)

95°C 15sec

60°C 30sec

.5°C/sec to 95°C