|
1. In a sterile 0.75 ml eppendorf tube prepare the
following mixture (depending on the number of RNAs to be reverse
transcribed).
|
Reagent
|
No. of
|
RNA
|
preps
|
|
|
| |
1
|
2
|
3
|
4
|
5
|
|
10 mM dNTPs
|
2.5*
|
5
|
7.5
|
10
|
12.5
|
|
5×RT Buffer (with DTT)
|
5
|
10
|
15
|
20
|
25
|
|
MMLV (200 U/µl)+
|
0.5
|
1
|
1.5
|
2
|
2.5
|
|
RNasin (3.3 U/µl)
|
1
|
2
|
3
|
4
|
5
|
|
DEPC water
|
1
|
2
|
3
|
4
|
5
|
|
Primer (25 pmol/µl)
|
1
|
2
|
3
|
4
|
5
|
|
Total
|
11
|
22
|
33
|
44
|
55
|
* amount in µl
+ Moloney Murine Leukaemia Virus reverse transcriptase
2. Add 11 µl of the mixture to 14 µl of each RNA template,
to make a total volume of 25 µl, and spin briefly in a microfuge.
3. Heat at 42°C for 1 h and then at 94°C for 10 min (either
in a waterbath or in the thermocycler).
4. Store at -20°C, and label clearly.
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