This is a cached page for the URL (http://axon.med.harvard.edu/~cepko/protocol/mike/R4.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Protocol.template

Protocol R.4

RT-PCR Analysis

Solutions

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

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.

• 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°.

pcr reaction

• 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.