Berglund:Post-Harvesting RNA

REVERSE TRANSCRIPTION

Every DNAsed sample will undergo a +RT and a –RT, therefore, for 6 wells, make enough gene specific primer master mix for 12 samples. For the +RT and –RT master mixes, make enough for 6 samples each.

Gene Specific Primer Master Mix For 1 rxn
Plasmid specific Reverse Primer (10uM) .1uL
10mM dNTPs .5uL
ddH2O 3.4uL
Total 4uL

+RT Master Mix For 1 rxn
5X 1st strand buffer 2uL
.1M DTT 1uL
Superscript III .25uL
ddH20 .75uL
Total 4uL

-RT Master Mix For 1 rxn
5X 1st strand buffer 2uL
0.1M DTT 1uL
ddH2O 1uL
Total 4uL

1. For each DNAsed RNA sample label 2 1.5mL eppendorf tubes +RT and –RT along with well name.
2. In the each tube add 2uL of DNAsed template and 4uL of the Gene Specific Primer Master Mix.
3. Incubate at 95C for 1 minute. Then quick put on ice for 1 minute. Centrifuge briefly.
4. To the +RT tubes add 4uL of the +RT Master Mix and to the –RT tubes add 4uL of the –RT Master Mix.
5. Incubate at 52C for 50 minutes.

PCR

The following PCR master mix was made:

PCR Master Mix For 1 rxn
10mM dNTP mix 0.4uL
10X Lab Taq Buffer 2uL
Fwd Primer (10uM) .7uL
Rev Primer (10uM) .7uL
ddH2O 13.5uL
Lab Taq .7uL
Total 20uL

The following PCR program was used:

1. 95C for 3 minutes
2. 95C for 30 seconds
3. 62C for 30 seconds
4. 72C for 2 minute
5. Go to 2 25 times
6. 72C for 5 minutes
7. 4C forever