This protocol is for RT-PCR of JAX project samples. The protocol has been optimized for these samples, but may be generalized as noted in several sections. The volume/tube in the table is calculated against 3.0 - 4.0µl of RNA. For example, for 7.5µl of RNA, multiply all variables by ≈2.
- 10X RT Buffer
- 25 mM MgCl2
- dNTPs Mixture (2.5 mM)
- oligo dT primer
- RNase Inhibitor (20 U/μL)
- MultiScribe Reverse Transcriptase (50 U/μL)
- Tissue Culture Grade DMSO
|Component||Volume/Tube (μL)||Final Concentration|
|RNase-free water||Variable to make total = 20 μL||--|
|10X RT Buffer||1.0||1X|
|25 mM MgCl2||2.2||5.5 mM|
|dNTPs Mixture (2.5 mM)||2.0||500 μM/dNTP|
|oligo dT primer||0.5||2.5 μM|
|RNase Inhibitor (20 U/μL)||0.2||1.25 U/L|
|MultiScribe Reverse Transcriptase (50 U/μL)||0.25||1.25 U/μL|
Note: If changing the reaction volume, make sure the final proportions are consistent with the recommended values above.
‡ Add Rnase free water to the total volume above to bring the total volume to 10µl.
§ Random Hexamers, oligo d(T)16, or sequence-specific reverse primers can be used for primers of cDNA synthesis.
Note: The RT reaction volume can vary from 10µL to 100µL. Increasing the RT reaction volume will reduce the total number of reactions.
- Make a mixture of H2O and RT buffer in the same eppendorf tube according to the table above.
- Add MgCl2 into the mixture in the eppendorf tube
- Add dNTP to the mixture in the eppendorf tube.
Note: Use the Oligo-dT and not the Hexamer.
- Add RNase Inhibitor to the mixture.
- Add the Reverse Transcriptase Enzyme (RT) into the mixture. Place mixture on ice.
- Use 3.0 - 4.0μL of RNA for the RT experiment as indicated above.
- Use a pipette or a repipetter to dispense between 6 - 7μL of mixture (cocktail) to the eppendorf tube containing the RNA to make a final volume of 10µl.
Note: For better mixture, fast spin in a Microcentrifuge for 5-10s
- Transfer the tubes to a Thermocycler. Set the Thermocycler using the RT pre-programmed, or set the Thermocycler according to the instructions in the table below:
|Time||10 min||30 min||5 min|
|Temperature||25 °C||48 °C||95 °C|
Note: It can be stored overnight at 4ºC.
Make PCR cocktail as follows:
Note: The volume prescribed for each reagent for the PCR cocktail is intended for 36 tubes or samples.
- Add dH2O (1.48 mL) to a 50 mL tube on ice.
- Add 550 μL of PCR buffer (from Taq Polymerase, PCR buffer, and MgCl2 kit). Store MgCl2 at 4°C.
- Add 275 μL of MgCl2 into tube on ice.
- Add (110 μL of dNTP mix). The dNTP is at a [100 μM]. Resuspend to [5µM]. Note: All four dNTPs are added into one (1) eppendorf tube.
- Make SYBR Green by diluting from 1000X into 100X in DMSO and H2O. 990 μL of sterile DMSO + 10 μL of SYBR Green. The working 200X diluted stock is prepared by making a 1:1 dilution of Cybergreen with dH2O (mix 500µl of Cybergreen into 500µl of H2O (1:1 dilution)).
- Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H2O, and MgCl2 cocktail on ice. Gently vortex. Keep on ice.
- Add 55 μL of Taq polymerase to the cocktail. Gently vortex, and keep on ice.
- Thaw frozen Primers kept in the -80ºC freezer, or make Primers. Primers arrive from IDT as lyophilized contents of a tube, they are then resuspended in sterile dH2O to a concentration of 0.1 nM/μL (this is the stock solution). The working solution is made by mixing 10 μL of primer with 90 μL dH2O. The final concentration of the Primers should be at 5µM.
- Take 10 μL of the forward (5' - 3') and reverse primers (3' - 5') of each gene of interest and add into the tubes containing the enzyme mix.
- Add 230 μL of PCR cocktail to each tube containing both the forward and the reverse primers.
- Align your PCR strip tubes.
- Add 60 μL of dH2O to 10 μL of the cDNA (RT reaction).
Note: Optimal cDNA dilution should be @ 1:6 fold dilution, thus if cDNA reactions were in 10 μL, add 60 μL dH2O.
- Add 80μL dH2O to the no template control strip tube.
- Spin each PCR tube down for 5 – 10s in a microcentrifuge.
- After spin, transfer the PCR mixture in each tube into the appropriate PCR strip tube, and cap.
- Place PCR tube in a rack. Keep on ice until analysis.
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare