NEC express5800 A2010b User Manual page 11

PrimeScript™ RT Master Mix （Perfect Real Time）
(2) After extraction of total RNA, remove genomic DNA by Recombinant DNase I
E. Confirmation of Genomic DNA Contamination
The presence of genomic DNA can be verified by real-time PCR without reverse
transcription. It is convenient to use a primer that can do PCR amplification from
both genomic DNA and mRNA for this experiment, and one tube should include
an exogenous genomic DNA aliquot . In addition, a primer which was designed
not to amplify genomic DNA could amplify derived from pseudogene. In this case,
this procedure can also be use to verify DNA.
URL:http://www.takara-bio.com
(RNase-free) (Cat. #2270A) treatment. After the reaction, DNase I should be
inactivated by either heat treatment or phenol/chloroform extraction.
Procedure
1. Prepare the following reagents :
total RNA
10X DNase I Buffer
RNase Inhibitor
DNase I (RNase-free)
DEPC treated water
2. Incubate at 37℃ for 20 min.
3. Perform one of the following procedures to inactivate DNase I
A. Heat treatment
(1) Add 2.5 μl of 0.5 M EDTA, incubate at 80℃ for 2 min.
(2) Increase reaction volume to 100 μl with DEPC treated water
B. Phenol/Chloroform extraction
(1) Mix 50 μl of DEPC treated water and 100 μl of phenol/
chloroform/isoamyl alcohol (25 : 24 : 1) together.
(2) Centrifuge at 15,000 rpm for 5 min. at room temperature
then transfer the upper layer to a new tube.
(3) Add equal amount of Chloroform/isoamyl alcohol
(24 : 1) and mix.
(4) Centrifuge at15,000 rpm for 5 min. at room temperature
then transfer the upper layer to new tube.
4. Add 10 μl of 3 M sodium acetate and 250 μl of cold ethanol, then
incubate on ice for 10 min.
5. Centrifuge at 15,000 rpm for 15 min. at 4℃ and remove the top clear
liquid.
6. Wash the precipitate with 70％ ethanol, and then centrifuge at
15,000 rpm for 5 min. at 4℃ and remove the supernatant.
7. Dry the precipitate.
8. Dissolve the precipitate in the proper amount of DEPC treated water.
Cat. #RR036Q
v201204Da_2
20 - 50 μg
5 μl
20 U
2 μl (10 U )
up to 50 μl
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