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