R334C and A107C

Elizabeth Thompson, Audrey Heathcote

R334C

Construct Sequence

PCR Mutagenesis 2/21/2022

Phosphorylation & Ligation 3/17/2022

Transformation 3/19/2022

Single Colony Inoculation (Miniprep) 3/25/22

Miniprep 3/26/22

Single Colony Inoculation (Midiprep) 4/6/22

Midiprep 4/7/22

Sequencing

A107C

Construct Sequence

PCR Mutagenesis 6/15/22

Phosphorylation & Ligation

Transformation

Single Colony Inoculation

Miniprep

Sequencing

Linearization

Transcription

A107C

PCR Mutagenesis A107C - 06/15/2022

Q5 Site-Directed Mutagenesis Kit - 25 uL Reaction Mix

*Probably do 2 tubes to balance throughout

MBG Water = 9.0 µL

Template DNA (25 ng/µL CFTR_pGHE in Box 6 Freezer C)) = 1.0 µL

10 µM (need to dilute stock!) A107C_CFTR_mut_FOR1 = 1.25 µL

10 µM (need to dilute stock!) A107C_CFTR_mut_REV1 = 1.25 µL

Q5 2X Master Mix (Box 12 Freezer C) = 12.5 µL

*Before cycling, ensure the PCR tubes do not have any air bubbles. If there are air bubbles present, tap the tubes on the table, and/or centrifuge them in the mini centrifuge. You may also flick them with your finger followed by spinning.

PCR Steps

Initial Denaturation: 98ºC for 30 seconds

25 Cycles: 98ºC for 10 seconds

52ºC* for 30 seconds

72ºC for 500 seconds (~8kB)**

Final Extension: 72ºC for 500 seconds (~8 minutes)

Hold: 4ºC

Cycling Conditions

*Annealing temperatures may vary depending on the primers being used.

**Extension time may need to be adjusted depending on the size of the plasmid. Assume a rate of 1 kb/minute to calculate a new time.

4. At the end of the reaction, place the tubes in the PCR Rack (Freezer A) if you’re not doing the next steps right away.

5. If you continue with the protocol, take 2 µL out of every PCR tube to prepare samples for gel electrophoresis analysis. If the reactions succeeded, draw a check mark on the side of the tube and continue with the next step. If the reaction failed, discard the tube in the trash and rethink the PCR strategy.

6. For the successful reactions, add 1 µL of DpnI (Enzyme Rack, Freezer A) to the remaining PCR product and digest at 37°C overnight. Write DpnI on the side of the tube. If in a rush, you may add 1 µL of FD DpnI and digest at 37°C for two hours.

Gel Electrophoresis - 06/15/2022

Agarose Gel Preparation (1% agarose gel)

NOTE: Ethidium bromide is a known mutagen and contact with it must be avoided. Wear gloves when handling it.

Weigh about 0.5 g of agarose (Chemical Shelves) and transfer into a 125 mL Erlenmeyer flask (Cabinet A).
Add 50 mL of 0.5X TBE (Cabinet A) to the Erlenmeyer flask containing the agarose. Gently swirl the flask to disperse the agarose evenly.
Heat for 30 seconds in the microwave, let cool down for 10 seconds, then heat for another 10 seconds.
Insert a thermometer (Drawer A) into the solution (don’t let it touch the bottom), wrap a KimWipe around the top to cover it, and let the agarose solution cool down to 70°C.
- Periodically swirl the flask to ensure the temperature is even throughout the solution! (every two minutes or so)
While waiting, obtain an electrophoresis tank and gel tray (both in Drawer A) wipe with Kimwipe and gently insert the gel tray in the tank in the casting position with the rubber gaskets touching the tank walls.
- Verify that the gaskets are not twisted out of the gel tray and they make a perfect seal with the tank on both sides.
When the agarose temperature nears 70°C, obtain a 10 µL pipette, and ethidium bromide (EtBr, Fridge A).
When the temperature is 70°C, add 2 µL of EtBr into the agarose solution.
- After pipetting the EtBr into the solution, set your pipette to 4 µL and flush the solution up and down to wash residual EtBr into the solution. Dispose of the pipette tip in the trash.
Pour the agarose solution into the gel tray and poke any air bubbles into a corner of the gel tray with a toothpick (Drawer A)
Insert the appropriate well comb (Drawer A) into the gel, then let it cool and solidify (it will turn opaque)

Gel Electrophoresis

Gather desired DNA samples. Write and number the names of the DNA according to their planned order in the gel.
Obtain as many 0.6 mL tubes as you have samples and label them. To each tube, add:
- 3 µL of MBG water (Fridge A)
- 2 µL of the DNA sample to its corresponding tube
- 1 µL of DNA dye (Fridge A)

NOTE: The water volume should be adjusted if more or less DNA volume is used. Remember to record the specific volume of DNA used.

~0.1-0.2 µg of DNA per 1 mm gel lane.

Check that the gel is solid by gently moving the tank around, then turn the gel around to where the rubber gaskets are not touching the tank walls.
Fill the electrophoresis tank with 0.5X TBE until the liquid level is the same on both sides of the tank and the gel is covered
Centrifuge the samples before placing them in their wells to mix the dye
Set the pipette to ~6.5 µL to make a complete transfer of the sample. Load samples according to the written order in the notebook. Do not pierce the gel.

NOTE: If a trail of dye floats to the surface while loading, don’t worry. The DNA should sink into the well.

NOTE: The DNA Ladder, whether QuickLoad or otherwise, must be kept on ice when not in use and promptly returned to the refrigerator afterwards.

Slide the tank cover on with the negative prod (black) on the same side as the DNA.
- Turn the electrophoresis machine on by flipping the switch on the front.
- Run the gel for 50 minutes at 140 V