TaqMan RT

DNA Free

1. In 26ul of sample, add 3ul of 10X Dnase I buffer and 1ul rDNase I
2. Incubate at 37°C for 20 min.
3. Add 3ul DNase Inactivation Reagent
4. Incubate 2 min at room temp with occasional mixing
5. Spin at 13,000 rpm for 1.5 min at room temperature.
6. Then transfer supernatant to a new tube.

TaqMan RT

1. Calculation the RNA we need in 8ul mix. O.D Conc.
   

   O.D. Conc.	Amount needed (1ug)	Water	Total
   e.g. 0.74ug/ul	1.35 ul	6.65 ul	8 ul

2. Master Mix
   

   	1x
   10x buffer	2ul
   MgCl2 buffer	4.4ul
   dNTPs	4ul
   Random Hexamas	1ul
   RNase Inhibitors	0.5ul
   Reverse Transcriptase	0.5ul
   Total	12.4ul

3. Mix the 8ul mix with the 12.4ul master mix together into little strip tube.
   (The total volume of the reaction is 20ul)
4. Run the reaction with rt-taqman program.
   (25°C for 10min ⇒ 48°C for 60min ⇒ 95°C for 5min ⇒ 4°C)

TaqMan-PCR

1. Mix the above solution into the little strip tube.
2. Run the reaction with tap-pcr program.
   (40 cycle, 50°C for 2 min ⇒ 95C for 15min ⇒ 95°C for 15sec ⇒ 61°C for 1min ⇒ 25°C for 60min)
3. Stop the reaction at 25°C.
4. Run in 2% gel and take picture.