半定量RT-PCR (Semi-Quantitative RT-PCR )
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Solutions
10X RT Buffer
10X PCR Buffer
100 mM Tris pH 9.0
500 mM KCl
1% Triton X-100
25 mM MgCl 2
use at a concentration of 1.5 mM
Lysis Solution
4M GuSCN 250 g guanidine thiocyanate
25mM Na citrate 7.0 17.6 ml 0.75M Na citrate pH 7.0
0.5% Sarkosyl 26.4 ml 10% Sarkosyl
add 293 ml Q
before use, add 72ml bME
Water Saturate Phenol
thaw 500 ml phenol
add 0.5 g hydroxyquinolin
add 500 ml Q
mix and allow phases to separate at room temperature
repeat 2 times and store at 4°C
3M NaOAc 5.2
24.6 g NaOAc (anhydrous)
pH to 5.2 with acetic acid
up to 100 ml Q
Procedure
rna isolation
• Add tissue to 400 microliters lysis solution with bME added just prior to use. If collecting several samples hold tubes on ice. These can be stored for years at -80°C.
• Add 1 microliter glycogen, 30 microliters 3M NaOAc 5.2 and 500 microliters water saturated phenol. Mix by gentle inversion and add 100 microliters chloroform.
• Mix by inversion and hold on ice for 15 minutes.