Biology Reference
In-Depth Information
1.
Remove culture medium, add 2.5 mL of Trizol™ reagent and lay the culture
flask flat so that all the cells are covered. Leave for 5 min then pipet up and down
several times using a 5-mL sterile pipet and transfer sample into microcentrifuge
tubes (1.25 mL into each tube). Add 250
L of chloroform to each tube, vortex
briefly then leave at room temperature for 5 min.
µ
2.
Centrifuge the samples for 10 min at 13,600 xg in a microcentrifuge. After cen-
trifugation transfer the upper aqueous layer to a fresh microcentrifuge tube. Add
500
L of isopropanol, mix by inverting the tube several times and incubate at
room temperature for 10 min. Pellet the RNA by centrifugation at 13,600 xg for
10 min. Carefully remove the isopropanol from the tube without disturbing the
pellet. Add 1 mL of 75% ethanol and respin. Carefully remove all traces of the
75% ethanol and allow the pellet to air-dry for 10 min.
µ
3.
Add 100
µ
L of DEPC-treated sterile H
2
O to each RNA sample and incubate at
50
°
C if necessary to resuspend the RNA pellet.
3.1.2. Isolation of Genomic DNA
There are many commercial “Kits” available for the purification of genomic
DNA from a variety of different sample sources, including blood, tissue and
cells. We have found that the spin column-based kits manufactured by
Qiagen are quick and easy to use and produce a good yield of high-quality
genomic DNA.
3.2. Reverse Transcription of mRNA
1.
Pipet 5
g of RNA into an microcentrifuge tube, add 50 ng of random hexamers
(pd(N)
6
) or 500 ng of oligo-dt primer and DEPC-treated sterile H
2
O to 12
µ
µ
L.
Incubate at 70
°
C for 10 min then place on ice for 2 min (
see
Note 2
).
2.
Add 2
µ
L of 10X RT buffer, 2
µ
L 25 m
M
MgCl
2
, 1
µ
L of 10 m
M
dNTP mix and
2
L of DTT, incubate the reaction for 5 min at room temperature when using
pd(N)
6
or 42
µ
°
C if using oligo-dt. Add 1
µ
L (200 U) of Superscript II RT enzyme
and incubate at 42
C for 1 h. (When using pd(N)
6
preincubate the reaction at
room temperature for 10 min prior to moving to 42
°
°
C.)
3.
Following incubation, terminate the reaction by incubation at 70
°
C for 5 min,
add 1
µ
L of RNase H, and incubate at 37
°
C for 30 min.
3.2.1. Amplification of Genomic DNA and cDNA by the PCR
1.
For amplification by PCR, use 5
µ
L of cDNA (from first strand reaction,
see
Note 2
) or 5
L reaction volume.
For each pair of oligonucleotide primers, prepare a “master mix” containing PCR
buffer, dNTPs, oligonucleotide primers and
Taq
DNA polymerase (
Tables 1-3
).
Mix 95
µ
L of genomic DNA (20-50 ng/
µ
L) in a 100
µ
L aliquots of this to 0.5-mL microcentrifuge tubes containing either
patient genomic DNA, cDNA, or appropriate control sample. Cover the reactions
with two drops of light mineral oil and amplify by PCR using the following 40
cycle parameters:
µ
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