Procedure- Site Directed PCR (with Q5 mix) and Dpn1

9.0 uL MBG H2O

1.0 uL CFTR_pGHE 25ng/uL (Freezer C, box 6)

1.25 uL E116C_CFTR_mut_FOR

1.25 uL E116C_CFTR_mut_REV

12.5 uL Q5 2X Master mix (Freezer C, box 12)

Annealed at 54ºC (2ºC lower than our lowest primer's MP)

PCR was successful (see E116C and R334C page for image).

1 uL of FD Dpn1 was added to both original PCR tubes (were not labeled precisely, so another gel will be run on both to determine which was the successful one) and left in incubator at 10:28 AM. Retrieved at ____.

Phosphorylation

1. Obtain a 0.6 mL tube and label it. Write CIRC below the DNA name add the reagents in the following order:

5 µL of MBG Water

2 µL of Linear PCR Product (PCR Rack, Freezer A)

1 µL DNA Ligase Buffer (Enzyme Rack, Freezer A) 

1 µL 10 mM ATP (Enzyme Rack, Freezer A) 

1 µL T4 PNK (Enzyme Rack, Freezer A) 

2. Incubate at 37ºC For 20 minutes. Write PHOS on the side of the tube. 

Ligation

1. In the same tube, add the reagents in the following order: 

6 µL MBG Water (Fridge A)

2 µL 50% PEG 4000 (Enzyme Rack, Freezer A)

1 µL T4 DNA Ligase Buffer (Enzyme Rack, Freezer A)

1 µL T4 DNA Ligase (Enzyme Rack, Freezer A)

2. Incubate at RT for one hour. Write LIG on the side of the tube. 

3. When done, place the tube in the freezer.

1. Determine how many LB Agar plates you need to make the correct amount of LB Agar. 20mL of LB Agar go on each petri dish. (Ex: 3.5g of LB Broth with Agar + 100mL of DI Water to make about 5 plates)

5 plates = 3.5 g LB Agar + 100 mL DI water

2. Measure out the determined amount of LB Broth with Agar (Chemical Shelves) using the analytical balance in the main lab.

3. Add the amount of DI water you need and then add the measured out LB Broth with Agar to a bottle.

4. Add a stir bar to the solution and stir/heat the agar on the magnetic stirring hot plate in the main lab. 

5. Remove the stir bar (using the magnetic rod). Move the bottle to the small autoclave in the shared equipment room (use the insulated gloves!).

6. Autoclave for 30 minutes at 121°C. 

7. Retrieve from autoclave and allow the bottle to cool to 65°C. Check with a thermometer usually found in the gel electrophoresis drawer.

8. Grab the desired amount of petri dishes from the main lab (in the back section) and bring them to the Tissue Culture Hood. Spray the hood with ethanol and wipe with a paper towel. Then open the culture plate bag and without touching the inside of the bag gently remove however many petri dishes you need. When done close the bag and return to the drawer in the main lab.

9. Retrieve the antibiotic corresponding to what bacterial DNA you are transforming (Ampicillin Box 5 in Freezer C).

10. Allow it to defrost and then add the desired amount to the cooled solution. 100 µL of antibiotic per 100 mL of solution. 

11. Swirl the bottle to mix and then pipette ~20mL of solution to each plate. Use the 25 mL pipettes in the main lab. 

12. Allow the plates to cool without the lids on for about 20 minutes in the tissue culture hood. Check that it is solid to ensure they are finished cooling.

13. Move to the Fridge B to store. Place the agar-side up (upside down), so condensation does not affect the LB Agar.

1. Add 5 µL of E116C DNA to a previously labeled 0.6 mL tube. Incubate on ice for 10 minutes.

2. Take the cooler with ice and go to the -80°C freezer. Obtain a tube of JM109 competent cells from the JM109 box (red tape). Place the tube on ice and let the cells defrost. While waiting, turn on the water bath and set at 42°C. Periodically check the water bath temperature and adjust the temperature dial accordingly. 

3. Add 10 µL of cells to the tubes containing circularized DNA and incubate on ice for 30 minutes. Record the total volume of cells removed from the tube. Subtract that volume from the volume written on the label. Replace the old label with a new one and write the new volume of cells in the tube. Replace the cells in the -80°C freezer.

4. Add 450 µL SOC media (Fridge B) to a previously labeled 2 mL tube and place it in the incubator. Turn on the small incubator/shaker in the equipment room down the hall.

5. Heat shock the cells in the 42°C water bath for 40 seconds and then let them rest on ice for 2 minutes. Transfer the cells to their corresponding tube with SOC media. Shake in the incubator at 170 rpm, 37°C for 1 hour. Place the culture plates (Fridge B) in the large incubator to dry.

6. After the incubation period, retrieve the cells and culture plates. Label the plates with the name of the DNA, your initials, and the date. Obtain a 100 mL beaker fill it about halfway with 95% ethanol (Fridge A) and ignite the Bunsen burner.

MN 07/15/2022

7. Place the culture plate on the rotating stand. Pipette all of the bacterial culture into the plate. Dip the metal spreader in the ethanol. Pass the spreader through the flame. Allow the flame to die and let the spreader cool for about 10 seconds. Spread the bacteria evenly on the plate. Replace the lid and remove the plate from the stand ensuring the agar side is down.

E116C/R104C

E116C

Negative Control (MBG Water)

8. Place the plates in the large incubator at 37°C and incubate overnight (16-24 hours).

Placed in incubator at 5:45 PM

16 hours: 9:45 AM

Taken out at ___ AM

Used 30uL JM109. 

When transferring heatshocked cells into SOC media, I spilled a little bit of the media. 

1. Obtain a bottle of LB Broth + Amp (100 µg/mL, Fridge A), the bacterial culture plates, a box of small pipette tips, and four 250 mL Erlenmeyer flasks (Cabinet A). Label the Erlenmeyer flasks with the names of the DNA. Take all the materials to the biological safety cabinet.

2. Rub ethanol on your gloves and allow it to dry. Pour 75 mL of broth into each Erlenmeyer flask. Obtain a pipette tip, remove the lid on one of the plates and visually inspect for an acceptable colony. Good colonies will be medium size and separate from other colonies. 

3. Once a colony is found, drag the pipette tip across the colony without touching other colonies. Ensure you have gotten as much of the colony as possible without getting another colony or scarring the agar. Drop the pipette tip straight down into its corresponding Erlenmeyer flask. Replace the lid on the plate and the foil on the flask.

4. After all flasks are inoculated, place them in the small incubator and shake at 37°C at 170 RPM for about 15-16 hours (until cloudy). Take all the materials back, clean the work area with ethanol, and lower the screen on the cabinet. Turn off the biological safety cabinet. 

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

NOTE: You must wear gloves to handle the bacterial culture. Gather TENS buffer, 3 M sodium acetate buffer (pH 5.2), vortex,  adaptors, 50 mL Falcon tubes, a 500 mL bottle, one 50 mL and one 10 mL graduated cylinder. All materials can be found in cabinet A. Place the materials on a cart. Label the Falcon tubes accordingly. Manipulation of PCI and chloroform must be done in the fume hood.

*Please note the first step is time sensitive make sure you are prepared before removing bacterial cultures from the shaker/incubator.

Removed from the incubator at 10:17am

1. Head to the large centrifuge room on the second floor with cart and bacterial cultures. Transfer 40 mL of the bacterial culture into a Falcon tube. Spin at 5,000 g for 3 minutes. Discard supernatant into waste jar. Repeat with the rest of the bacterial culture in the same tube.

2. Add about 15 mL of TENS buffer. Vortex for 10 seconds or until the pellet is dissolved.

3. Add about 7.5 mL of 3 M sodium acetate pH 5.2. Vortex for 10 seconds.

4. Centrifuge for 5 minutes at 10,000 g and transfer supernatant to a new, previously labeled Falcon tube.

5. Add 10 µL of RNase (Enzyme Rack, Freezer A) and wait for 20 minutes. 

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

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

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. 

9. Carefully decant supernatant into the ethanol waste bottle. Add 40 mL of 70% ethanol (Fridge A) and centrifuge at 10,000 g for 2 minutes. 

10. Carefully decant supernatant in the ethanol waste bottle and let DNA dry. Draw a circle on the outside of the tube where the DNA pellet is. Leave the tubes uncapped and place a Kimwipe over the top.

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 to 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 1 mL of MBG water (Fridge B), vortex, and transfer to a previously labeled 1.5 mL tube.

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

• Nanodrop results: 2507.47ng/uL

• Sequencing File

50 mL Reaction Mix (go in order)

X uL MBG water to 50 uL

X uL DNA (30-50 ug)

5 uL 10X Buffer (Enzyme Rack, Freezer A)

1 uL Restriction Enzyme NheI (10-20 units, Enzyme Rack, Freezer A)

*Place all in 1.5 mL tube labeled S108C LIN and incubate overnight at 37*C to completely digest, then move to temporary rack in Freezer A

PCI Extraction

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 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.

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.

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).

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. 

FORWARD: 24.0 nmol = 240 µL of MBG water for 100 uM stock solution

REVERSE: 32.1 nmol = 321 µL of MBG water for 100 uM stock solution

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

FORWARD: S108C_CFTR_mut_FOR1 100 µM

REVERSE: S108C_CFTR_mut_REV1 100 µM

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

Store in Freezer C

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!) S108C_CFTR_mut_FOR1 = 1.25 µL 

10 µM (need to dilute stock!) S108C_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: 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 (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 (PCR Product)

   • 1 µL of DNA dye (Fridge A) 

3. Fill the electrophoresis tank with recycled 0.5X TBE (Cabinet A) until the liquid level is the same on both sides of the tank and the gel is covered  

4. Add 5 uL of DNA Ladder to first well (box in Freezer A)

5. 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.

7. Slide the tank cover on with the negative prod (black) on the same side as the DNA. 

   • Run the gel for 50 minutes at 140 V

Procedure- Site Directed PCR (with Q5 mix) and Dpn1

9.0 uL MBG H2O

1.0 uL CFTR_pGHE 40 ng/µL (Freezer C, box 6)

1.25 uL E116C_CFTR_mut_FOR

1.25 uL E116C_CFTR_mut_REV

12.5 uL Q5 2X Master mix (Freezer C, box 12)

Annealed at 54ºC (2ºC lower than our lowest primer's MP, should have done 2-3ºC higher than lowest MP)