Linearization

VIII. DNA Linearization (Video)

A. Reaction Setup

1. In the lab notebook, copy the list of reagents from the box below. 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.

0.1 ng/uL --> 5140 ng/uL /1000 = 5.140 ug

50/5.14 = 9.7 uL of 0.1 ng/uL KCNJ6

1.0 ng/uL --> 3337 ng/uL /1000 = 3.337 ug

50/3.337 ug = 14.98 uL of 1.0 ng/uL KCNJ6

10 ng/uL KCNJ6 --> 9507 ng/uL /1000 = 9.507 ug

50/9.507 ug = 5.26 uL of 10 ng/ul KCNJ6

Xho1 restriction enzyme & 10x FastDigest Buffer

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.

Labeled KCNJ6 __ ng/uL LIN

Placed in Incubator at 9:35 am 3/29/22

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.

B. Phenol:Chloroform Extraction of Linearized DNA

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

Obtain & Label 6 new 1.5 mL tubes for intermediate steps.

1. If the reaction volume was 50 µL, 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. (Machine in back of lab)

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. (Transferred ~ 95 uL of top layer from each tube.) Add 100 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute. (Back of Lab) (We accidentally used a benchtop centrifuge instead of the vortex, which means that our solution may not have mixed as well as we needed.)

4. Transfer top layer to a new, previously labeled 1.5 mL tube. (Took out ~50 uL of top layer from each 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. (repeat next day and continue to step 5)

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. (A pellet only showed in 1.0 ng/uL tube. Leaving to sit overnight and will repeat tomorrow)

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.

Let dry overnight (Set 0.1 ng/uL and 1.0 ng/uL tubes out to dry at 5:30 pm 3/30. 10 ng/uL tube still showed no pellet)

7. Transfer phenol and chloroform wastes from all tubes into the appropriate waste bottle. (In fume hood)

8. Re-suspend the dried DNA in 10 µL of MBG water (Fridge A). (Can start transcription immediately after this). Thursday afternoon.