RT-PCR using Superscript III
Part I: First-Strand cDNA Synthesis
Note: remember to spin down reagent tubes as they may contain small volumes! Also note that
many of these reagents have similar names (for example, RNA, RNAseOUT, RNAseH, or DTT,
Oligo(dT), dNTP, etc.). Pay attention and don’t mix them up!
1.
Combine the following in a 1.5ml microcentrifuge tube:
8μl
RNA (your RNA, or borrow from a friend; dilute if over 1μg / μl)
1μl
50μM oligo(dT)20
1μl
10mM dNTP mix
2.
Denature the RNA by incubating at 65°C in a water bath for 5 minutes
3.
Place on ice for at least 1 minute to anneal the oligo(dT) to the poly(A) tail of the
mRNAs
4.
Prepare cDNA synthesis mix in a new tube
2μl
10x RT buffer
4μl
25mM MgCl2
2μl
0.1 M DTT
1μl
RNaseOUT
1μl
SuperScript III RT
5.
Add 10μl of cDNA Synthesis Mix to the RNA/primer mix, mix gently, centrifuge
briefly
6.
Reverse translate the RNA to DNA by incubating for 50 minutes at 50°C
7.
Terminate the reaction by incubating at 85°C for 5 minutes
8.
Chill on ice to cool
9.
Centrifuge briefly. Add 1μl RNase H.
10.
Remove RNA by incubating for 20 minutes at 37°C
Part II: Amplification of target DNA
1.
2.
Combine the following in a PCR tube
2μl
cDNA (the tube from part 1, end of Step 10)
10μl
2X Brilliant QPCR Master Mix with low ROX
1μl
primer mix (contains both forward and reverse)
1μl
SYBR green 1 (already diluted 1/1000)
6μl
H20
Spin down and hand in to TA to amplify and quantify cDNA in a real-time PCR
instrument