BISC311:DNA amplification

Step 1 mRNA isolation (see wing disc protocol) Trizol extraction. MRNA extracted and diluted in 13ul of DEPC water @-80C

Step 2 DNAse treatment Materials: 	PROMEGA kit 70-80% Ethanol DEPC water 3M sodium acetate (pH 5.2) Isopropanol

•	30 min at 37C •	COOL and SPIN •	Add 2uL of DNAse stop solution •	10 min at 65C •	COOL and SPIN

•	To 20ul of sample, add 10 % volume (2 µl) of 3 M sodium acetate (pH 5.2) and either same amount (20 µl) of Isopropanol.

•	Keep at –20ºC for at least 1hr.

•	Centrifuge (14000 rpm, 10 min)

•	Remove supernatant, and rinse pellet with 70-80 % ethanol.

•	Centrifuge (14000 rpm, 10 min)

•	Remove supernatant and air dry pellet (10-15 min).

•	Dissolve into 5-10 µl DEPC water. Step 3 Spec

cDNA synthesis Materials: 	Fermentas cDNA synthesis kit

1ug (X uL)	RNA 1uL 	dT primer 11uL 	Total (if not add DEPC up to 11ul •	Mix and spin for 3-5 sec •	70C for 5min 4uL 	5X reaction buffer 1uL 	Nuclease inhibitor 2uL 	10mM dNTP mix •	Mix and spin •	37C for 5min 2uL 	Rev transcriptase •	37C for 1hr •	70C for 10min 20uL 	Total

Store at –20C

Step 4 PCR Materials: 	PROMEGA DoTaq Polymerase TBE Buffer Gel Primers dNTPs

•	Vortex <5s Taq PCR Master Mix.

Component Volume/reaction Make MM Taq PCR buffer	5 Distilled water (provided)	15.875 dNTPs	0.5 Primer fw (10µM)	0.5 Primer rv (10µM)	0.5 MgCl2	2 Taq polymerase	0.125 Template DNA	0.5 Total volume	25

PCR reaction - Initial hold for 2 minutes at 94 degrees C to melt template - X cycles: •	melt template: 30s @ 94 degrees •	anneal primers: 30s @ X degrees (depends on Tm of primer pair) use Tm-5C •	extension: X min@ 72 degrees (approx 1 min / kb) - final extension: 5 minutes @ 72 degrees - 4C forever

•	Analyze products by agarose gel electrophoresis o	Run gel with TBE buffer and 1.5% agarose. 100mV for ~30 min.

Step 5 Gel extraction: Use MinElute kit to purify band. Stored in H2O at –20C. Materials: 	MinElute kit from Qiagen