I. In vitro transcription
In a sterile 1.5 ml microcentrifuge tube, combine the following:
4 ul 5 times transcription buffer
2 ul 0.1 M DTT
4 ul 2.5 MM NTP(A, C, G)
0.8 ul RNase inhibitor (25 U/ul) RNase inhibitor (Promega)
100 UM cold UTP
100 UM cold UTP
1 ul/ug UL linear DNA template
5 ul 10 UCI / UL P32 UTP (800 Ci/mmol)
1 ul RNA polymerase SP6, T7 or T3
The total volume is ~20 ul.
Incubate for 1 hour at 37 °C.
2 ul of each transcribed DNase I, incubated for 20 minutes at 37 °C.
Second, the probe purification
The purification probe uses QIAGEN's QIAquick Nucleotide Removal Kit or Boehringer Spin Column (G50 Sephadex). Check 1 ul in the flashing counter, P32 channel. A good probe will be 5 x 10 5 ~ 1 x 10 6 CPM.
Third, hybrid
Open the heatblock to 95C. For samples in water or ethanol, dry the appropriate amount of RNA and include a tube with 1 ul of tRNA or glycogen (Sigma), which serves as a negative control.
Each sample should have the following:
24 ul formamide
2 ul 0.6 M tube
2.4 ul 5 M sodium chloride
0.3 ul 0.1 M EDTA
2×10 5 cpm probe
5×10 4 cpm load control probe
DEPC water
DEPC water
Total volume 30ul
And mix the samples. Heat at 95 ° C for 10 minutes. Incubate for 4 hours at 55 °C.
Fourth, RNase digestion
Ribonuclease digestion buffer:
300 MM sodium acetate
10 mM TRIS
5 mM EDTA
Add 350 ul of digestion buffer to each sample.
1 ul 4 mg/ml ribonuclease A and 0.4 ul 10 u/ul ribonuclease T1.
Incubate, 30 ° C, 45 minutes to 1 hour.
5. Proteinase K
Incubate 20% SDS and 2.5 ul of 10 mg/ml proteinase K into each sample for 15-20 minutes at 10 °C at 37 °C.
Six: clean up
Extract 400 ul phenol/chloroform/isoamyl alcohol (25:24:1)
Transfer the supernatant to a new tube. Add 1000 ul of 100% ethanol and 1 μl of 10 mg/ml glycogen. Mix well.
Samples were incubated at -70 ° C for 30 minutes or in a dry ice / ETOH bath for 10 minutes.
Centrifuge for 15 minutes. Aspirate EtOH. Let the ball dry in the air.
Reload the dye in 8 ul of formamide. Allow to sit at RT for 5-10 minutes, often stirring.
Seven: Polyacrylamide gel electrophoresis analysis
Hot sample for 5 minutes at 100 °C. A molecular weight marker loaded to 5% polyacrylamide/7 M urea denatured gel 2000 cpm. Run the gel 38-42 mA. Dry gel, exposed to membrane O / N in a -70C intensifying screen.
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