Shreffler:RTPCR: Difference between revisions
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==Overview== | ==Overview== | ||
This is for RT-PCR of JAX project samples. | 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== | ==Materials== | ||
* | * 10X RT Buffer | ||
* | * 25 mM MgCl<sub>2</sub> | ||
*5 | * dNTPs Mixture (2.5 mM) | ||
* oligo dT primer | |||
* RNase Inhibitor (20 U/μL) | |||
* MultiScribe Reverse Transcriptase (50 U/μL) | |||
* Tissue Culture Grade DMSO | |||
==Procedure== | ==Procedure== | ||
# | {| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:710px" <!-- | ||
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#Make ' | | align="center" style="background:#f0f0f0;"|'''Component''' | ||
# | | align="center" style="background:#f0f0f0;"|'''Volume/Tube (μL)''' | ||
# | | align="center" style="background:#f0f0f0;"|'''Final Concentration''' | ||
# | <!-- Copy and paste one of the lines above to create a new column in the schedule table. Alternatively, you can also delete lines to reduce the number of columns.--> | ||
# | |-- | ||
# | | RNase-free water | ||
| Variable to make total = 20 μL | |||
| -- | |||
|-- | |||
| 10X RT Buffer | |||
| 1.0 | |||
| 1X | |||
|-- | |||
| 25 mM MgCl<sub>2</sub> | |||
| 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 | |||
|-- | |||
| '''Total''' | |||
| '''≈6.15/tube''' | |||
| -- | |||
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.--> | |||
|} | |||
'''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 H<sub>2</sub>O and RT buffer in the same eppendorf tube according to the table above. | |||
# Add MgCl<sub>2</sub> 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: | |||
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:710px" <!-- | |||
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| align="center" style="background:#f0f0f0;"|'''Step''' | |||
| align="center" style="background:#f0f0f0;"|'''Incubation''' | |||
| align="center" style="background:#f0f0f0;"|'''RT''' | |||
| align="center" style="background:#f0f0f0;"|'''Inactivation''' | |||
<!-- Copy and paste one of the lines above to create a new column in the schedule table. Alternatively, you can also delete lines to reduce the number of columns.--> | |||
|-- | |||
| style="background:#f0f0f0;"|'''Time''' | |||
| 10 min | |||
| 30 min | |||
| 5 min | |||
|-- | |||
| style="background:#f0f0f0;"|'''Temperature''' | |||
| 25 °C | |||
| 48 °C | |||
| 95 °C | |||
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.--> | |||
|} | |||
'''Note: It can be stored overnight at 4ºC.''' | |||
'''Make PCR cocktail as follows:''' | |||
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:710px" <!-- | |||
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| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''100X''' | |||
| align="center" style="background:#f0f0f0;"|'''550X''' | |||
<!-- Copy and paste one of the lines above to create a new column in the schedule table. Alternatively, you can also delete lines to reduce the number of columns.--> | |||
|-- | |||
| PCR Buffer | |||
| 100 | |||
| 550 | |||
|-- | |||
| MgCl<sub>2</sub> | |||
| 50 | |||
| 275 | |||
|-- | |||
| dNTPs | |||
| 10 | |||
| 110 | |||
|-- | |||
| Primers | |||
| 10+10 | |||
| -> | |||
|-- | |||
| SYBR | |||
| 5 | |||
| 55 | |||
|-- | |||
| Taq | |||
| 5 | |||
| 55 | |||
|-- | |||
| dH<sub>2</sub>OPrimers | |||
| 270 | |||
| 1485 | |||
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.--> | |||
|} | |||
'''Note: The volume prescribed for each reagent for the PCR cocktail is intended for 36 tubes or samples.''' | |||
# 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 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.''' | |||
# 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 making a 1:1 dilution of Cybergreen with dH<sub>2</sub>O (mix 500µl of Cybergreen into 500µl of H2O (1:1 dilution)). | |||
# 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. 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 (this is the stock solution). The working solution is made by mixing 10 μL of primer with 90 μL dH<sub>2</sub>O. '''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 dH<sub>2</sub>O 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 dH<sub>2</sub>O.''' | |||
# Add 80μL dH<sub>2</sub>O 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== | ==Discussion== | ||
Line 26: | Line 189: | ||
==References== | ==References== | ||
<biblio> | <biblio> | ||
# | # ref1 pmid= | ||
</biblio> | </biblio> | ||
Latest revision as of 14:25, 14 October 2009
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
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 |
Total | ≈6.15/tube | -- |
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:
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 |
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.
Discussion
References
-
pmid=
Contact
- Who has experience with this protocol?
- Email Wayne Shreffler through OpenWetWare