Shreffler:RTPCR

Overview
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.

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
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.'''


 * 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.

Note: Use the Oligo-dT and not the Hexamer.


 * 1) Add RNase Inhibitor to the mixture.
 * 2) Add the Reverse Transcriptase Enzyme (RT) into the mixture.  Place mixture on ice.
 * 3) Use 3.0 - 4.0μL of RNA for the RT experiment as indicated above.
 * 4) 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


 * 1) 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:

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.


 * 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 making a 1:1 dilution of Cybergreen with dH2O (mix 500µl of Cybergreen into 500µl of H2O (1:1 dilution)).
 * 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.  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 (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.
 * 9) 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.
 * 10)  Add 230 μL of PCR cocktail to each tube containing both the forward and the reverse primers.
 * 11)  Align your PCR strip tubes.
 * 12)  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.


 * 1)  Add 80μL 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
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Contact

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