RT-PCR experimental steps.

,
tissue
pumping:
1. Take tissue block 50-100mg with liquid nitrogen ground into powder in the research
2. Transfer into the glass homogenizer to add 1 ml Trizol pumping slurry
3.Move into 1.5ml new
centrifugal tube
5 ml needle pumping
4.ice incubation for 5 min
5.centrifugation 12,000g 4 oC 5 min Take liquid transfer into 1.5ml new centrifugal tube
6.plus 0.2 2 ml chloroform, concussion, incubation on ice for 5 min
7.centrifugation<12,000g 4 oC 10 min Take the upper liquid phase into 1.5 ml new centrifuge tube
8.plus 0.5 ml isopropyl alcohol, shock, incubation on ice Breeding 5 min
9.centrifugation< 12,000g 4 oC 5 min Discard the liquid
10.Add 1 ml 75% ethanol, shock
11.centrifugation<7,500g 4 oC for 5 minutes. Discard the liquid
12. Make it transparent at room temperature
13. Add DECC treated water 10 sl -- 35 sl dissolved RNA (can be stored in liquid nitrogen or
low temperature refrigerator
)
14. deRNA denaturation electrophoresis observation 18s, 28s strip,
photophosometer
detection 260/280 suction Luminosity ratio, calculation of RNA concentration
2, RT reaction system (first step):
about 20 minutes
RNA extract
5 μl
Radome quote
2 μl
RNA sin
0.5 μl
1.65 oC 15 min
2. Immediately put in the ice bath
.