1. Locate your primers in the package and ensure they are the primers you ordered.

2. Compare the nucleotide sequences and GC% on each primer label to the order invoice. The melting temperature will likely differ between the label and the order (this is not important).

3. Spin down the tubes using the centrifuge machine. SPIN ALL TUBES BEFORE OPENING! This is because dry pellet can often come dislodged during shipping and could be in the cap.

4. Create a 100uM master stock by adding MBG water into the tube. Take the amount of nanomoles (nmols) on the label and multiply it by 10. That number is how many microliters of MBG water you'll add to the tube.

Added 289 uL of MBG water to L218C_CFTR_mut_FOR1 primer.

Added 269 uL of MBG water to L218C_CFTR_mut_REV1 primer.

5. Label the top of each primer tube with its name and concentration (100 uM).

Labeled "L218C 100 uM For1" and "L218C 100 uM Rev1"

6. Wait 10 miuntes to allow the master stock to rest at room temperature and then mix well (centrifuge) before creating the working solutions.

Stored in CFTR True-Open box in Freezer C.

Followed the protocol for Site-Directed Mutagenesis PCR

9 uL of MBG Water (fridge a)

1 uL of 25ng/uL of CFTR_pGHE (box 6, freezer c)

1.25 uL of 10uM of L218C_CFTR_mut_FOR1 primer (CFTR True-Open box, freezer c)

1.25 uL of 10uM of L218C_CFTR_mut_REV1 primer (CFTR True-Open box, freezer c)

12.5 uL of Q5 mastermix (box 12, freezer c)

Annealing temperature changed to 65C

Extension time changed to 500 seconds/8 minutes

Two tubes were created. L218C-1 and L218C-2

Removed 2 uL from tube 1 and 2uL from tube 2. Placed in freezer a. 1 uL of FD Dpn1 was added to each original PCR tube and left in incubator at 1:02pm. Retrieved and placed in freezer at 9:58am the following morning.

L218C_CFTR_pGHE-1: 644.0 ng/uL

L218C_CFTR_pGHE-2: 649.0 ng/uL

Phosphorylation and Ligation completed by protocol

L218C PCR Dpn1 tube 2 was used

L218C CIRC tube left in freezer A

Made 10 plates per protocol

1238.5 ng/uL

Dilute to 100 ng/uL, 10 ng/uL, and 1 ng/uL concentrations +water negative control

Transformation ran per protocol

Added 40 uL of JM109 Cells

Plates left in Ingrid (37C) at 11:20am

Transfer cultures into 14 mL Falcon tubes and spin at 5,000 g for 3 minutes. Discard supernatant into waste jar.

2. Add about 2 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

Added 4 mL

3. Add about 1 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 3 minutes at 5,000 g and transfer supernatant to new, previously labeled Falcon tube.

5. Add 2.5 µL of RNase (Enzyme Rack, Freezer A) and wait for 5 minutes.

6. Add about 1 mL of PCl (Fridge A) from the bottom layer. Vortex for 10 seconds and centrifuge at 5,000 g for 5 minutes.

7. Transfer the top layer to a new, previously labeled Falcon tube. Add about 1 mL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 5,000 g for 3 minutes.

8. Transfer the top layer to a new, previously labeled Falcon tube. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 5,000 g for 5 minutes.

No pellet. Add 700 uL and sodium acetate. Left overnight!

9. Carefully decant supernatant into the ethanol waste bottle. Add 5 mL of 70% ethanol (Fridge A) and centrifuge at 5,000 g for 2 minutes.

Two large pellets were seen and are drying overnight.

10. Carefully decant supernatant into the ethanol waste bottle and let DNA dry.

Left overnight to dry

11. Transfer phenol and chloroform wastes from all tubes into the appropriate waste bottle.

*Waste jar and bacterial culture flasks from step 1 will need to be autoclaved.

*Falcon tubes that contained TENS buffer and sodium acetate from steps 3-4 can be discarded in the trash.

*Falcon tubes that contained phenol and chloroform need remain in the fume hood until dry. Once dry they can be discarded in the trash. Steps 6-7

*Pipette tips from phenol and chloroform steps need to be placed in tip waste beaker inside of fume hood. Tips need to be disposed of when dry.

12. Re-suspend the dry DNA in 20 µL of MBG water (Fridge A), vortex, and transfer to a previously labeled 1.5 mL tube.

Resuspended in 40 uL MBG Water and placed in freezer A. The 14 mL falcon tubes were placed in freezer A as well.

L218C_CFTR_pGHE_1 : 30290.2 ng/uL

L218C_CFTR_pGHE_2 : 30952.6 ng/uL

Sequencing

Results: Correct mutation of TTG --> TGC

Forward: resuspended 240 uL MBG

Reverse: resuspended 321 uL MBG

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!) L100C_CFTR_mut_FOR1 = 1.25 µL

10 µM (need to dilute stock!) L100C_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.

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.

1. Weigh about 0.5 g of agarose (Chemical Shelves) and transfer into a 125 mL Erlenmeyer flask (Cabinet A).

2. 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.

3. Heat for 30 seconds in the microwave, let cool down for 10 seconds, then heat for another 10 seconds.

4. 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)

5. 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.

6. When the agarose temperature nears 70°C, obtain a 10 µL pipette, and ethidium bromide (EtBr, Fridge A).

7. 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.

8. 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)

9. Insert the appropriate well comb (Drawer A) into the gel, then let it cool and solidify (it will turn opaque)

Gel Electrophoresis

1. Gather desired DNA samples. Write and number the names of the DNA according to their planned order in the gel.

2. 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.

3. 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.

4. 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

5. Centrifuge the samples before placing them in their wells to mix the dye

6. 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.

7. 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

Q5 Site-Directed Mutagenesis Kit - 25 uL Reaction Mix

*Probably do 2 tubes to balance throughout

MBG Water = 9.0 µL

Template DNA (L218C_1 (30290.2 ng/µL diluted to 25 ng/µL) in Freezer A) = 1.0 µL

10 µM (need to dilute stock!) L100C_CFTR_mut_FOR1 = 1.25 µL

10 µM (need to dilute stock!) L100C_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

59ºC for 30 seconds

72ºC for 500 seconds (~8kB)**

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

Hold: 14ºC

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 (box in the door, 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.

3 µL MBG water

2 µL PCR product

1 µL of dye

Ladder: 5 µL

Samples: 6 µL

50 minutes at 150 V

Nanodrop:

L218C/L100C-1 : 892.9 ng/uL

L218C/L100C-2 : 752.7 ng/uL

Ran a gel to confirm it was circularized. Redid it 8-14-22

• Used 30uL JM109 cells

Placed in the incubator at 6:48 pm, will remove around 9:48am - 10:48am

Removed from the incubator at 10:17 am.

The pellet was an adequate size, but I wanted to precipitate more. I added 1/10 total volume of 100% ethanol and let it sit overnight.

• Did not run a gel first, nanodrop showed the presence of dsDNA.

• Nanodrop results:

  • • L218C/L100C - 1 : 4317.73 ng/uL

    • L218C/L100C - 2 :

• Sequencing File