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IX. RNA Synthesis: In Vitro Transcription
NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated.
A. mMESSAGE mMACHINE T7 Ultra Kit (Freezer B)
1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Add the reagents in the following order and incubate at 37°C for 2 hours. Add a tally mark on the box.
*if needed you can stop after this step. Make sure to place samples in Freezer A for temporary storage.
B. Turbo DNAse (Part of the same kit, Freezer B)
1. Add 1 μL of Turbo DNase to the RNA tube(s) and incubate at 37 °C for 15 min.
2. Take out Poly(A) Tailing reagents to thaw.
C. Poly(A) Tailing (Part of the same kit, Freezer B)
1. Add the following reagents into the RNA tube(s) and incubate at 37°C for 1 hour.
**NOTE: Do NOT use regular MBG water from Fridge A! Either use what is in the kit or if there is not any in the kit, use the RNA MBG water in Freezer C Box 13.
2. After Poly(A) Tailing incubation, immediately add 10 μL ammonium acetate stop solution (part of the same kit, Freezer B) to RNA tubes and mix by pipetting.
*Samples can be placed in Freezer A for temporary storage.
D. Phenol:Chloroform Cleanup of RNA
1. Add 110 µL of acidic PCl (pH 4.5, Fridge B) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.
2. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 g for 1 minutes.
3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of isopropanol (Fridge A) and place in -20°C freezer (any normal freezer) for 30 minutes.
4. Centrifuge the tubes for 15 minutes at 13,000 rpm and 4°C. Use the centrifuge in the cold room on the 3rd floor.
5. Carefully decant supernatant down the drain by tilting the tube(s) until it is completely upside down. Add 500 µL of 70% ethanol (Fridge A) and centrifuge at 13,000 rpm for 1 minute.
6. Carefully decant supernatant down the drain by tilting the tube(s) until it is completely upside down. Touch the tube to a Kimwipe to wick away any residual ethanol. Set the tube(s) on a rack, open the cap, and set a Kimwipe on top.
7. Transfer phenol and chloroform wastes from all tubes into the appropriate waste bottle.
8. Re-suspend the dry RNA in 15 µL of MBG water (part of the same RNA kit, Freezer B).
Please put the RNA tube in an appropriate box in the -80 freezer (2nd floor).
Other RNA Synthesis Kits
Other RNA Synthesis Kits
**DO NOT USE THIS UNLESS SPECIFICALLY ASKED TO DO SO
HighYield T7 ARCA mRNA Synthesis Kit FOR N1-methylpseudo-UTP
NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated.
Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Based on the reagent list below, determine ahead of time how much template DNA will be required for your reaction. The amount of PCR-grade water added depends on the volume of linearized template DNA necessary.
Math: The total volume is 20 µL and you want your final DNA mass to be between 1-10 µg. You can do M1V1=M2V2 to determine the amount of your linearized DNA template to add based on the mass of your linearized DNA.
i.e. (20 µL)(5 µg) = (X µL)(### µg)
Retrieve the HighYield T7 ARCA mRNA Synthesis Kit from Freezer B. Add a tally mark on the box.
Place HighYield T7 RNA Polymerase Mix on ice.
Thaw all other reagents to RT, then vortex and spin down.
Add the following reagents to your labeled 1.5 mL tube in order:
X µL PCR-grade water (based on amount of Template DNA required to get to 20 µL; determine the amount of template DNA required before beginning RNA synthesis)
2 µL HighYield T7 Reaction Buffer 10X
2 µL DTT
Vortex and spin down
1.2 µL ARCA
0.3 µL GTP
1.5 µL N1-Methylpseudo-UTP **this is what is different from the normal T7 ARCA mRNA kit
1.5 µL CTP
1.5 µL ATP
X µL Linearized Template DNA
Vortex and spin down
2 µL HighYield T7 RNA Polymerase Mix
Vortex and spin down
TOTAL VOLUME: 20 µL
Incubate at 37ºC for two hours.
When complete, you can freeze in Freezer A for temporary storage.
PolyA Tailing (different kit in Freezer B)
1. Add the following reagents into the RNA tube(s) and incubate at 37°C for 1 hour.
**NOTE: Do NOT use regular MBG water from Fridge A! Either use what is in the kit or if there is not any in the kit, use the RNA MBG water in Freezer C Box 13.
36 µL Nuclease Free Water
20 µL 5X E-PAP Buffer
10 µL 25 mM MnCl2
10 µL ATP Solution
4 µL E-PAP Enzyme
Incubate at 37ºC for 1 hour.
2. When complete, you can freeze in the -80ºC Freezer on the 2nd floor.
**DO NOT USE THIS UNLESS SPECIFICALLY ASKED TO DO SO
HighYield T7 ARCA mRNA Synthesis Kit
NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated.
Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Retrieve the HighYield T7 ARCA mRNA Synthesis Kit from Freezer B. Add a tally mark on the box.
Place HighYield T7 RNA Polymerase Mix on ice.
Thaw all other reagents to RT, then vortex and spin down.
Add the following reagents to your labeled 1.5 mL tube in order:
X µL PCR-grade water (based on amount of Template DNA required to get to 20 µL; determine the amount of template DNA required before beginning RNA synthesis)
2 µL HighYield T7 Reaction Buffer 10X
2 µL DTT
Vortex and spin down
1.2 µL ARCA
0.3 µL GTP
1.5 µL UTP
1.5 µL CTP
1.5 µL ATP
X µL Template DNA (want 1-8 µg DNA)
Vortex and spin down
2 µL HighYield T7 RNA Polymerase Mix
Vortex and spin down
TOTAL VOLUME: 20 µL
Incubate at 37ºC for two hours.
When complete, you can freeze in Freezer A for temporary storage.
PolyA Tailing (different kit in Freezer B)
1. Add the following reagents into the RNA tube(s) and incubate at 37°C for 1 hour.
**NOTE: Do NOT use regular MBG water from Fridge A! Either use what is in the kit or if there is not any in the kit, use the RNA MBG water in Freezer C Box 13.
36 µL Nuclease Free Water
20 µL 5X E-PAP Buffer
10 µL 25 mM MnCl2
10 µL ATP Solution
4 µL E-PAP Enzyme
Incubate at 37ºC for 1 hour.
2. When complete, you can freeze in the -80ºC Freezer on the 2nd floor.
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