Transcription

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. (Red box in Freezer B)

Labeled KCNJ6 __ ng/uL RNA.

Added reagents below as listed (used the big red box in Freezer B) & put in incubator at 3:50 pm 3/31/22

*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. 

put in incubator at 5:54 pm on 3/31/22

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.

(Used NF (nuclease-Free) water instead of MBG water)

Put in incubator at 6:18 pm on 3/31/22

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 mix by inverting 3 times. Place the mixtures in -20°C freezer (any normal freezer) for 30 minutes.

Put in the freezer at 3:25 pm on 4/1/22

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. (stairs by norimatsu's office, go into cold room, all the way in the back on the left)

Put in centrifuge at 3:55 pm --> very small pellets formed, put in Freezer A to recentrifuge on Sunday & see if a bigger pellet forms.

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).