BISC311:DNA amplification

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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

Solution Amount
RNA 13uL
10X buffer 2uL
DNAse 5 uL

• 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