10X RT Buffer
10X PCR Buffer
100 mM Tris pH 9.0
500 mM KCl
1% Triton X-100
25 mM MgCl2
use at a concentration of 1.5 mM
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
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.
Spin for 10 minutes at 4°C and remove the aqueous phase. Add 500 microliters isopropanol, hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
Resuspend the pellet in 300 ml Lysis Solution and add 300 ml isopropanol. Hold at -20°C for 1 hour and spin at 4°C for 30 minutes. Wash and dry.
Resuspend the pellet in 10 ml DEPC treated Q and store at -80°C.
reverse transcription reaction
Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml).
Heat to 65° for four minutes and immediately transfer to ice.
Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:
2 microliters 10X RT buffer
1 microliter 1 mg/ml BSA
1 microliter 20 mM DTT
0.5 microliters RNaseIN
0.4 microliters 25 mM dNTPs
0.2 microliters AMV RT
9 microliters Q
Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°.
Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction.
Add the following PCR cocktail per tube:
2.5 microliters 10X PCR buffer
1.5 microliters 25 mM MgCl2
1 microliter each primer (10pmol/microliter; 55° Tm)
0.2 microliters 25 mM dNTPs
0.025 microliters a-32P dATP
0.02 microliters Taq polymerase (protocol T.2)
14 microliters Q
Perform PCR using the following cycle parameters:
94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes.
The number of cycle should be determined empirically to find the linear range of amplification.