Question: You are performing a reverse transcription (RT) reactions for high-throughput analysis using a 96-well plate format. Each 96-well plate is 8 horizontal rows of wells by 12 vertical columns of wells. You need to set up the entire 96-well plate will identical RT components except for the particular primers that you will be using. Each of the 8 columns on the plate will get it’s own unique primer pair. The best way to accomplish this is to produce a 1 giant mastermix containing all the necessary components minus the primers, and then aliquot the primers as needed into the 8 columns. RT reactions require the following components: buffer, template RNA, dNTPs, RT polymerase, primers, and water. The final volume of each RT reaction is 25 ul.
Reagents available:
2X RT Buffer
50 uM dNTPs
537 ug/ul RNA
1.0 units/ul RNA polymerase
Primer mix at 100 uM
Sterile water
Based on your protocol, each RT reaction requires: 1X RT buffer, 5 uM dNTPs, 50 ng/ul RNA, 0.5 units of RT polymerase, and 10 uM each primer.
(A)How much of each component will you need for 1 RT reaction? Show your work
(B)How much of each component will you need for a mastermix for all 96 reactions? Show your work.