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Reverse Transcription Procedure for Two-Step RT-PCR:
Add the following reagents to a thin-walled 200 or 500 ml PCR microcentrifuge tube on ice:
Volume Reagent Final Concentration
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5 ml RNA template 10 pg/ml (0.05-0.25mg/ml total RNA or desired Ploy A RNA
1 ml Deoxynucleotide mix 500 mm each dNTP
1 ml Control primers 1 mm each
-or-
Random nonamers 2.5 mm
-or-
Specific primer ------
-or-
Oligo (dT)12-18 3.5 mm
7.5 ml Water ------
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14.5 ml Total Volume
Note: The oligo (dT)12-18 cannot be used with the RNA template that does not have a polyadenylated tail such as Ribosome RNA.
Mix gently and briefly centrifuge to collect all components to the bottom of the tube.
Place tube in thermal cycler between 70 °C and 85 °C for 10 minutes.
Remove tube, place on ice, centrifuge and add the following components to the reaction:
Volume Reagent Final Concentration
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4 ml 5 x buffer for AMV-RT 1 x
1 ml Enhanced avian RT 1 U/ml
0.5 ml RNase inhibitor 1 U/ml
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20 ml Total Volume
Incubate the reaction tubes at 25 °C for 15 minutes if using random nonamers or oligo (dT)12-18. This pre-incubation step allows these primers to be extended by the enhanced avian RT before incubating at 42°C.
Place tubes at 42° C for 50 minutes.
Note: Raising the transcription reaction temperature incrementally (up to 65 °C) is recommended for transcribing templates with difficult secondary structure. If the transcription reaction is run at elevated temperatures, a drop in yield may occur.
The first strand of cDNA is now ready for subsequent PCR amplification, cloning, library synthesis, etc.
Plasmid DNA purification
2D SDS PAGE
siRNA transfection
siRNA design principles
Microplate reader