×
Please leave your comments or questions
 
cDNA Synthesis and qPCR
Author: Erin M Green, updated date: , view: 23, Q&A: 0

Procedure

cDNA synthesis

Follow protocol from Biorad iScript reverse transcriptase (described below).

 

In 8-strip PCR tubes, mix the following reagents for each reaction:

(include controls with no RT)

 

5x iSript reaction buffer      4 μl

RNA                            1 μg total

Reverse transcriptase        1 μl

RNAse-free water              (volume to 20 μl total)

 

In PCR machine, run “cDNA” program 

 

25ºC, 5 min

42ºC, 30 min

85ºC, 5 min

4ºC, 10min

 

Store cDNA at -20ºC. 

 

qPCR

Set up master mixes for 10 µL reactions as follows:

(For total amount of master mix, multiply each by total number of reactions plus two. Perform each reaction in triplicate).                                             

Mix#1:

2x SYBR Green mix                       5 µL                

20 µM forward gene-specific primer 0.25 µL

20 µM reverse gene-specific primer 0.25 µL

 

Mix#2

cDNA                                0.5 µL

Water                                4 µL

 

Combine reactions in 384 well plate, adding Mix#1 to wells first, followed by Mix#2.

 

In qPCR machine, run 2-step protocol with melt curve

      95ºC for 3 min

Repeat for 40 cycles:

      95ºC for 10 sec

      55ºC for 30 sec

Melt curve:

55-95ºC, 0.5ºC increments


Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.