Shreffler:RTPCR

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# Add 275 μL of MgCl<sub>2</sub> into tube on ice.
# 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 H<sub>2</sub>O.  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.
-
# Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H<sub>2</sub>OH2O, and MgCl<sub>2</sub> cocktail on ice.  Gently vortex.  Keep on ice.
+
# Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H<sub>2</sub>O, and MgCl<sub>2</sub> 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.
# 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 dH<sub>2</sub>O 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 dH<sub>2</sub>O.  
# 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 dH<sub>2</sub>O 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 dH<sub>2</sub>O.  

Revision as of 17:27, 3 June 2009

Contents

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.


  1. Make a mixture of H2O and RT buffer in the same eppendorf tube according to the table above.
  2. Add MgCl2 into the mixture in the eppendorf tube
  3. Add dNTP to the mixture in the eppendorf tube.
  4. Add RNase Inhibitor to the mixture.
  5. Add the Reverse Transcriptase Enzyme (RT) into the mixture. Place mixture on ice.
  6. Use 7 μL of RNA for the RT experiment.
  7. 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

  1. 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
  1. Add dH2O (1.48 mL) to a 50 mL tube on ice.
  2. Add 550 μL of PCR buffer (from Taq Polymerase, PCR buffer, and MgCl2 kit). Store MgCl2 at 4°C.
  3. Add 275 μL of MgCl2 into tube on ice.
  4. 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.
  5. 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.
  6. Add 55 μL of 200X diluted SYBR Green to the PCR buffer, H2O, and MgCl2 cocktail on ice. Gently vortex. Keep on ice.
  7. Add 55 μL of Taq polymerase to the cocktail to the mixture on ice. Gently vortex, and keep on ice.
  8. 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.
  9. Take 10 μL of the forward and reverse primers and add into the tube containing the enzyme mix.
  10. Add 230 μL of PCR cocktail to each tube containing both primers.
  11. Align your PCR strip tubes.
  12. 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.

  1. Add 140 μL dH2O to the no template control strip tube.
  2. Spin each PCR tube down for 5 – 10s in a microcentrifuge.
  3. After spin, transfer the PCR mixture in each tube into the appropriate PCR strip tube, and cap.
  4. Place PCR tube in a rack, keep on ice until analysis.

Discussion

discuss this protocol

References

  1. pmid= [ref1]


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