Making first-strand cDNA
Mix the following ingredients in a pcr tube and use a pcr machine as a heat block. Alternatively,
use a 1.5 ul tube and heat blocks containing water and/or water baths.
1.
2.
3.
4.
5.
6.
7.
8.
___ul RNA
1 ul of poly-dT primer,
FLE94
Incubate at 70 C for 5
minutes
Place tube on ice, add:
10 ul of reverse
transcriptase buffer
2 ul of dNTPs
___ul water
1 ul of MMLV-Reverse
transcriptase enzyme
9. Incubate at 42 C for 1 hr
10. Incubate at 90 C for 5
min.
11. Store cDNA at -20 C
1 ug total RNA
0.5 ug/ul stock
To allow primer
annealing
-5X stock; vortex stock
to mix well
25.0 mM stock
To give total reaction
volume of 50 ul,
including enzyme.
Gently mix reaction;
quick spin if
necessary.
To make cDNA
To kill enzymes
Long-term storage at 80 C