Shreffler:RTPCR: Difference between revisions
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# Add dH<sub>2</sub>O (1.48 mL) to a 50 mL tube on ice. | # Add dH<sub>2</sub>O (1.48 mL) to a 50 mL tube on ice. | ||
# Add 550 μL of PCR buffer (from Taq Polymerase, PCR buffer, and MgCl<sub>2</sub> kit). Store MgCl<sub>2</sub> at 4°C. | # Add 550 μL of PCR buffer (from Taq Polymerase, PCR buffer, and MgCl<sub>2</sub> kit). Store MgCl<sub>2</sub> at 4°C. | ||
# Add 275 μL of | # Add 275 μL of MgCl<sub>2</sub> 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. | # 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 mixing 1:1 with dH<sub>2</sub>O. | # 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 mixing 1:1 with dH<sub>2</sub>O. |
Revision as of 14:23, 3 June 2009
Overview
This 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.
Materials
- 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
Procedure
Component | Volume/Tube (μL) | Final Concentration |
RNase-free water | Variable to make total = 20 μL | -- |
10X RT Buffer | 2.0 | 1X |
RNA | 7.0 | nanogram range |
25 mM MgCl2 | 4.4 | 5.5 mM |
dNTPs Mixture (2.5 mM) | 4.0 | 500 μM/dNTP |
oligo dT primer | 1.0 | 2.5 μM |
RNase Inhibitor (20 U/μL) | 0.4 | 1.25 U/L |
MultiScribe Reverse Transcriptase (50 U/μL) | 0.5 | 1.25 U/μL |
Total | 20 | -- |
Note: If changing the reaction volume, make sure the final proportions are consistent with the recommended values above.
- 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.
- Add RNase Inhibitor to the mixture.
- Add the Reverse Transcriptase Enzyme (RT) into the mixture. Place mixture on ice.
- Use 7 μL of RNA for the RT experiment.
- Use a pipette or a repipetter to dispense 7 μL of mixture to the eppendorf tube containing the RNA
Note: For better mixture, fast spin in a Microcentrifuge for 5-10s
- Transfer the tubes to a Thermocycler (17-60). Set the Thermocycler using the RT pre-programmed by Sasha, or set the Thermocycler according to the table below:
Step | Incubation | RT | Inactivation |
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:
' | 100X | 550X |
PCR Buffer | 100 | 550 |
MgCl2 | 50 | 275 |
dNTPs | 10 | 110 |
Primers | 10+10 | -> |
SYBR | 5 | 55 |
Taq | 5 | 55 |
dH2OPrimers | 270 | 1485 |
- 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 mixing 1:1 with dH2O.
- Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H2OH2O, and MgCl2 cocktail on ice. Gently vortex. Keep on ice.
- Add 55 μL of Taq polymerase to the cocktail to the mixture on ice. 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. That is the stock solution; the working solution is made by mixing 10 μL of primer with 90 μL dH2O.
- Take 10 μL of the forward and reverse primers and add into the tube containing the enzyme mix.
- Add 230 μL of PCR cocktail to each tube containing both primers.
- Align your PCR strip tubes.
- Add 120 μL of dH2O to 20 μL of the cDNA (RT reaction).
Note: Optimal cDNA dilution is 1:6, thus if cDNA reactions were in 10 μL, add 60 μL dH2O instead.
- Add 140 μL 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.
Discussion
References
-
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Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare