Realtime PCR protocol
1. Reverse Transcription
Reverse Transcription is carried out with the M-MLV Reverse Transcriptase
(Promega, M1701) and Random Primers (Promega, C1181):
1. Prepare the following RNA/primer mixture in each tube:
Total RNA
1-2 g
random hexamers (0.5ug/l)
1 l
DEPC H2O
to 15 l
2. Incubate the samples at 70°C for 5 min and then on ice for at least 1 min.
3. Prepare reaction master mixture. For each reaction:
M-MLV 5X Reaction Buffer 5 l
10 mM dNTP
1.25 l
M-MLV RT (200units / l) 1 l
DEPC H2O
2.75 l
4. Add the reaction mixture to the RNA/primer mixture (final volume is 25 l),
mix briefly.
5. Incubate the tubes at 37°C for 60 min.
6. 1:4 dilute 1st strand cDNA product with dd H2O (add 100l dd H2O to 25l
RT product), and then store at -20°C until use for real-time PCR.
2. Real-time PCR
1. Normalize the primer concentrations and mix gene-specific forward and
reverse primer pair. Each primer (forward or reverse) concentration in the
mixture is 5 .
2. Set up the experiment (Don’t forget to add detectors and select all the
wells!) and the following PCR program (default protocol) on ABI Prism SDS
7000. Select correct reaction volume (35 or 40l) and then click on the
dissociation protocol.
1) 50°C 2 min, 1 cycle
1
2) 95°C 10 min, 1 cycle
3) 95 °C 15 s -> 60 °C 1min, 40 cycles
4) 60°C 20 min, 1 cycle
3. A real-time PCR reaction mixture can be either 35 l or 40 l. Prepare the
following mixture in each optical tube. Do two replications for each reaction.
17.5 l SYBR Green PCR Mix* (2x)
20 l SYBR Green PCR Mix (2x)
4 l diluted cDNA or DNA (5-50ng)
4 l diluted cDNA or DNA
OR 3 l primer pair mix (5  each
3 l primer pair mix (5 M each
primer)
primer)
10.5 l H2O
13 l H2O
* Strongly recommend preparation of a Master Mix except different
template samples or primer pair mix. You may load Master Mix first and
then load different template samples or primer pair mix into each well.
4. After PCR is finished, analyze the real-time PCR result with the SDS 7000
software. Check to see if there is any bimodal dissociation curve or abnormal
amplification plot.
Note:
* Preparation of SYBR Green PCR Mix (2x):
qPCR Core kit for SYBR® Green I, 500rxns, (Eurogentec,
RT-SN73-05)
Component
Volume
10x Reaction Buffer
2.5ml
50mM MgCl2 solution
1.75ml
5mM dNTP solution
1ml
Hot GoldStar enzyme (5U/µl)
125µl
1/2000 dilution (in DMSO)of Sybr Green I stock
750µl
Water
6.375ml Total Mix
12.5ml
Aliquot and store at -20°C.
You can also use pre-made 2x master mix: SYBR Green PCR master mix, 200
reactions (Applied Biosystems) Or qPCR MasterMix Plus for SYBR® green I
w/o UNG (Eurogentec, RT-SN2X-03WOU+).
2