4-5-22 -- 4-6-22

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. Used concentration 2 because it had the largest pellet. Due to time constraints, we did not run a gel on the linearized products.

*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. See Protocol page for reagent list

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. Placed in freezer A at 5:15pm on 4-4-22

D. Phenol:Chloroform Cleanup of RNA Began at 6:15pm on 4-5-22

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.

Our pellet was not obvious after the first 15 minutes, so we centrifuged another 5 minutes. This was the result after centrifuging the second time. There was a layering of the solution, which might suggest chloroform contamination. We are continuing with step 5 in hopes that our sample is not contaminated. If it is, we will repeat transcription with one of our other three concentrations we linearized.

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).

4-6-22 at 4:06 PM Nanodrop concentration of RNA:

Concentration 1: 2127.4 ng/µL

Concentration 2: 1814.1 ng/µL

Concentration 3: 1809.2 ng/µL

Average Concentration: 1916.9 ng/µL

After determining RNA concentration with the Nanodrop, tube was placed in -80ºC freezer in box labeled "Extra RNA" in slot 40. A gel may be run on this RNA to determine whether it is pure or not.