RT-PCR protocol (real time pcr)
REVERSE TRANSCRIPTION PCR
PROTOCOL (23-3-2009)
DNAse TREATMENT:
20
ul extracted RNA+2.5 ul DNAse turbo
buffer 10X+1 ul Turbo DNAse (final vol 23.5 ul)
1.
Incubate at 37° for 30 min. Add 1 ul DNAse more. Incubate for 30’ again.
2.
Stop with the inactivation reagent. Centrifuge 1-2 min at maximum rpm and recover
supernatant.
RT:
1.
Quantify
DNAse- treated RNA with Nanodrop. Use 1 ug RNA for RT. Calculate the RNA volume
(ul) necessary.
2.
Bring
the RNA to the final volume of 35 ml with sterile water.
3.
Add
3 ml of 50 mM random hexamers to each sample.
4.
Incubate
10 min at RT.
5.
Add
19.5 ml of RT mix for each tube.
Prepare mastermix in 10% excess.
RT MIX FOR
EACH SAMPLE
Buffer
RT (5X) 11.5 ml
DTT
0.1 M 2 ml
dNTPS
(10 mM) 2 ml (100 mM stock)
RNAse
inhibitor 0.5 ml
RT
enzyme 1 ml
Water 2.5 ml
Final
volume= 35+3+19.5=57.5 ml
6.
37ºC
incubation for 1 hr 30´. To stop the RT reaction, heat at 70-75 ºC for 10´.
7.
Store
at -20ºC.
Q-PCR TAQMAN
FOR EACH
SAMPLE
Mastermix- 12.5 ml
F
primer- 1.25 ml
R
primer- 1.25 ml
tRNA-
2.5
Probe-
1.25
H2O-
3.75 ml
Sample
cDNA/ standard plasmid dilutions- 2.5 ml
Absloute
quantification and relative quantification using the standard curve.
THE END:)
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http://workspace.office.live.com/#akedarg/Documents/trcLentiVirusInfect.pdfThanks to the original creator of the file. I am just a collector. For some protocols, i am the writer.
If the protocols are useful, do comment on my guestbook.
Thanks,
Kedar Ghimire