Rt-PCR help,come in - use RT (Aug/09/2002 )
I am a fresh one in trying RT. I use b-actin as control to compare different GENE. I think both primers should be put into the same tube. But At the first time we use 25 cycles,we could find b-actin stripe. After we change some characters, we could not find it at last. Could you give me a better protocol, or even a little advise on playing with RT-PCR? Thanks a lot.
I don't what protocol you use for RT-PCR.I recommand the use of the superscript II reverse transcriptase from life technologies and following the supplied protocol. If the lengh of your PCR Product is important remove the ARN with RNAse H after the reverse transcription . For the PCR try some differents annhiling temperature (55,60,65°C for example. The time for elongation must be approximatively one minut per Kb (3 minuts for a PCR products of 3Kb). The Mg2+ concentration is also important try different: concentrations from 1 to 2mM for exemple. Put only 2µl(maximum) of your RT product into the PCR
RT PCR PROTOCOL 1
USING SUPERSCRIPT REVERSE TRANSCRIPTASE
1£®First Strand cDNA Synthesis:Add the following components to a nuclease-free microcentrifuge tube,total reaction volume is 20ul:
random primers:1ul 50-250 ng
Total RNA: 1-5 ug
DEPC-treated water to 12 ul
Heat mixture to 70¡æ for 10 min and quick chill on ice.
Collect the contents of the tube by brief centrifugation and add:
Components adding volume Final concentration
5X First Strand Buffer : 4ul 1mM
0.1 M DTT 2ul 10mM
10 mM dNTP 1ul 0.5mM
2£® Mix contents of the tube gently and incubate at 42¡æ for 2 min.
3£®Add 1 ul (200 units) of SUPERSCRIPT II, mix by pipetting gently up and down.
4. Incubation at 25¡æ for 10 min
5. Incubate 50 min at 42¡æ.
6. Inactivate the reaction by heating at 70¡æ for 15 min.
NOTE: The cDNA can now be used as a template for amplification in PCR. However, amplification of some PCR targets (those >1 kb) may require the removal of RNA complementary to the cDNA. To remove RNA complementary to the cDNA, add 1 ul (2 units) of E. coli RNase H and incubate 37¡æ for 20 min.
II. PCR Reaction
Use only 10% of the first strand reaction for PCR. Adding larger amounts of the first strand reaction may not increase amplification and may result in decreased amounts of PCR product.
1. Add the following to a PCR reaction tube for a final reaction volume of 100 ul:
Components adding volume Final cincentration
10X PCR Buffer 10ul 1mM
50 mM MgCl2 3ul 1.5mM
10 mM dNTP 2ul 0.2mM
Primer 1 (10 uM) 2ul 0.2mM
Primer 2 (10 uM) 2ul 0.2mM
Taq DNA polymerase (2-5 U/ul) 1ul
2 ul cDNA (from first strand reaction, preferably RNase H-treated)
Autoclaved, distilled water 80ul
2. Mix gently .
3. Perform 15 to 40 cycles of PCR.