Linearization

April 4, 2022

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.

Concentration 1: (40ug DNA)(1000ng/ug)(1uL/5140ng) = 7.78uL DNA, 36.22 uL MBG H2O

Concentration 2: (40ug)(1000ng/ug)(1uL/3337ng) = 12.00uL DNA, 32.00 uL MBG H2O

Concentration 3: (40ug)(1000ng/ug)(1uL/4507ng) = 4.21uL DNA, 39.79 uL MBG H20

Plasmid: pcDNA3, used Xho1 Restriction Enzyme

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 PCl and chloroform.

1. Add 50 µL of MBG water (Fridge A).

2. Add 100 µL of PCl (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. We centrifuged again without adding any extra sodium acetate or ethanol.

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. Small pellets were seen with each concentration, so we decided to do this step to see if we could precipitate more DNA.

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. Because the DNA pellet was not securely on the wall of the tube, we used a micropipette to remove the supernatant.

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.

Let dry overnight or for up to 2 days.

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).

Tubes with final linearized product are in Freezer A in tubes labeled 1, 2, and 3 next to the other KCNJ5 DNA.