mRNA VS Modified mRNA

VIII. DNA Linearization (Video)

A. Reaction Setup

1. In the lab notebook, copy the list of reagents from the box below following the format for a notebook entry for this protocol. Using the concentration of your sample calculate the volume of DNA solution needed to obtain your target mass of DNA (typically between 30-50μg).  The water volume will be determined by how much is needed for the reaction mixture to reach 50 µL.

NOTE: If a volume of DNA greater than 44 µL is needed, you must do a 100 µL reaction. To do that, simply double the amount of enzyme and buffer used and adjust the water volume to ensure the final reaction is 100 µL.

2. Obtain a 1.5 mL tube and label with the name of the DNA plus LIN. Add the reagents following the order in the table above. Incubate overnight at 37 °C to ensure complete digestion. Move to temporary rack in freezer A.

B. Phenol:Chloroform Extraction of Linearized DNA

NOTE: You must wear gloves and work under the fume hood when handling the PCI and chloroform.

1. If the reaction volume was 100 µL, skip this step. If the reaction volume was 50 µL, add 50 µL of MBG water (Fridge A).

2. Add 100 µL of PCI (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 100 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

4. Transfer top layer to a new, previously labeled 1.5 mL tube. Add 10 µL of 3M sodium acetate buffer (pH 5.2, Cabinet A). Add 220 µL of 100% ethanol (Fridge A). Mix by inverting 10 times and centrifuge at 13,000 rpm for 5 minutes.

NOTE: If no pellet is seen, add 20 µL 3 M sodium acetate buffer (pH 5.2) and spin again at 13,000 rpm for 5 minutes. If that does not work, let the DNA sit overnight and resume the protocol the following day by re- inverting the tube and repeating the centrifugation.

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 dried DNA in 10 µL of MBG water (Fridge A).

1. Add 110 µL of acidic PCI (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 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.

Let it sit overnight and spun again the next day

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 and placed in COVID-19 Project Box in -20C Freezer