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)

10 plates = 7 g LB Agar + 200 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.

(Choose the size of the bottle based on how much solution you're making. Ensure there is room in your bottle, so it won't overflow. Ex: If you're making 200mL of LB Agar, you would use a 500mL bottle.)

4. Add a stir bar to the solution and stir/heat the agar on the magnetic stirring hot plate in the main lab. Set the stirring setting as high as it can go where the stir bar is spinning in the middle without clanking the sides. Heat to boiling (about medium heat) and allow the powder to dissolve.

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. Use a sharpie to label the plates with "LB + Amp" initials and date on the edge of the plate. 

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

11. Allow it to defrost and then add the desired amount to the cooled solution. 100 µL of antibiotic per 100 mL of solution. Ex: 500uL of spectinomycin for 500mL of LB Agar solution.

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

13. 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.

14. Move to the Fridge B to store. Place the agar side up, so condensation does not affect the LB Agar.

1. Add 5 µL of R104C/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.

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.

8. Place the plates in the large incubator at 37°C and incubate overnight.

Placed in incubator at 4:13PM on 06/03/2022

Removed and placed in Fridge B at 1:20PM on 06/04/2022

Negative Control: No bacterial cultures present, indicating a sterile procedure. 

62 ng/uL: Bacterial growth present, but they are small and very concentrated. 

6.2 ng/uL: Bacterial growth present and the colonies are sufficiently sized and nicely spaced, but upon closer inspection, they are not round, indicating that multiple colonies have grown together. They also seem a little too small and   concentrated towards the edge of the plate. 

These issues could have been caused by incorrect reporting of concentrations or too long of an incubation period. We will test both options (diluting the 6.2 ng/uL sample and a shorter incubation period) at a later date.

We made another concentration (1ng/uL) of DNA because both concentrations gave colonies that were too concentrated. It was transformed 06/06/2022. When removed from the incubator on 06/07/2022, there was only one colony present. We are not sure why this happened, but it will be enough for single colony inoculation. We will do single colony inoculation and maxiprep with all 3 concentrations. 

Protocol:

1. Obtain a bottle of LB Broth + Amp (100 µg/mL, Fridge A) (add 1uL of Amp for every mL LB Broth), 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. 

Put in the incubator at 5:21 pm. Removed at 9:00 am. All of the flasks with bacterial cultures in them were cloudy, indicating proper colony growth. The negative control was not cloudy, indicating a sterile procedure.

A. Maxiprep

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.

*Steps 1-4 can be done in the centrifuge room on the second floor. 

1. Head to the large centrifuge room on the second floor with cart, waste jar, yellow tube holders, 3 falcon tubes (Labeled), a tube rack, the key, a pen, 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. Go back upstairs

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.

Go back downstairs with all of the above stuff and 3 new falcon tubes

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

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

6. Add about 5 mL of PCl (Fridge B) (use the big boy micropipette, do in fume hood and discard in proper place) 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.          Audrey messed up here and ended up discarding the top layer for the 1ng/uL and 6.2 ng/uL. There was some left though, so we switched to a midiprep at this point.

Put old falcon tube in PCI waste. Add about 1 mL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 5,000 g for 3 minutes. 

Added 1/10 total volume of sodium acetate buffer (200 µL for each) and left it to sit overnight. 

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

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-suspended the dry DNA in 2 mL of MBG water (Fridge B), vortex, and transfer to a previously labeled 1.5 mL tube.

The protocol calls for resuspension in 1 mL of MBG water for maxiprep; the DNA is twice as dilute as it should have been. 

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: 

   • 4 µL of MBG water (Fridge A) 

   • 1 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 40 minutes at 140 V

E116C+R104C_CFTR_1 : 55.8 ng/uL

E116C+R104C_CFTR_2 : 75.2 ng/uL

These concentrations are way too low because the DNA was resuspended in too much water; will need to be restarted from Maxiprep.

Primers

Results

1. Add an equal volume of 100% ethanol (Fridge A) and centrifuge at 5,000 g for 5 minutes.

Did this twice

No pellet for 62 ng/uL DNA, small pellet for 1 ng/uL DNA. Added 1/10 total volume of sodium acetate buffer (375 uL for each) and left it to sit overnight. 

Did two more times; still not very good pellets but they exist

2. Carefully decant supernatant into the ethanol waste bottle. Add 5 mL of 70% ethanol (Fridge A) and centrifuge at 10,000 g for 2 minutes. 

3. 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.

7/12/22

4. Re-suspend the dry DNA in 50 µL of MBG water (Fridge B), vortex, and transfer to a previously labeled 1.5 mL tube.

7/13/22

4 µL MBG water

1 µL DNA

1 µL dye

____________________

5 µL GeneRuler 1kb DNA Ladder in well

50 minutes at 150 V

E116C/R104C 1 ng/µL

1: 35.0 ng/µL

2: 35.2 ng/µL

E116C/R104C 62 ng/µL

1: 109.7 ng/µL

2: 105.9 ng/µL

3: 106.1 ng/µL

Concentrations are still too low to linearize with. This double mutant will have to be re-transformed to start over maxiprep completely.

Sequencing

R104C correctly mutated from AGA --> TGT

E116C correctly mutated from GAA --> TGC

This plasmid was made a few years ago and also has the inconsistency, which makes me think that it is actually a problem with the construct sequence of the plasmid and not a problem with the PCR mutagenesis procedure or any mutagenesis primers. 

1. Add 5 µL of R104C/E116C 62 ng/µL DNA to a previously labeled 0.6 mL tube. Incubate on ice for 10 minutes. While waiting, turn on the water bath and set at 42°C. Periodically check the water bath temperature and adjust the temperature dial accordingly (turn to ~58ºC). 

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. 

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. 

6. 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.

7. 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

8. 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)

9. Place the plates in the large incubator at 37°C and incubate overnight (agar side down).

Placed in incubator at 5:45 PM

16 hours: 9:45 AM

Taken out at 10:35 AM

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. 

Negative Control only had about 50 mL broth

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. 

Finished at: 6 PM

15 hours: 9 AM

16 hours: 10 AM

Retrieved at 9:50 AM 

Negative control was successful (liquid was still translucent) and all three experimental samples were also successful (cloudy).

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.

*Steps 1-4 can be done in the centrifuge room on the second floor. 

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.

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. 

Added 1/10 total volume of sodium acetate buffer (4 mL for each)  to 1 and 3 and left it to sit overnight. 

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. 

*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.

Successful products now in Box 21 in Freezer C for storage

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: 

   • 4 µL of MBG water (Fridge A) 

   • 1 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 45 minutes at 150 V

Sample 1 Average: 39.87 ng/µL

Sample 2 Average: 2094.2 ng/µL

Sample 3 Average: 24.9 ng/µL

1. 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.

2. Add reagents: 

27 µL MBG water

17 µL DNA (35.6 µg DNA)

5 µL 10X FD buffer (enzyme rack, FreezerA)

1 µL FD Nhe1 restriction enzyme **(incubate overnight for Nhe1, but for FD Nhe1 (FastDigest), do 2 hours)

3. 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 for 2 hours at 37°C to ensure complete digestion. Move to temporary rack in freezer A.

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.

Had to stop 07/19/2022 because the centrifuge refused to work for me. Resumed 07/20/2022.

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). 

5. 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.

6. 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. 

7. 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. 

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

9. Re-suspend the dried DNA in 10 µL of MBG water (Fridge A).

Held in Box 21 in Freezer C. 

RNA Synthesis: In Vitro Transcription

NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated. 

A. mMESSAGE mMACHINE T7 Ultra Kit (Freezer B)

1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Add the reagents in the following order and incubate at 37°C for 2 hours. Add a tally mark on the box.

mMessage mMACHINE T7 Ultra Kit

 10 µL T7 2X NTP/ARCA

 2 µL 10X T7 Reaction Buffer

 6 µL Linear DNA template

 2 µL T7 Enzyme Mix

Incubate for 2 hours at 37ºC

**Can freeze for temporary storage 

B. Turbo DNAse (same kit in freezer B) 

1. Add 1 µL Turbo DNAse to the RNA tube(s) and incubate at 37ºC for 15 min. 

C. Poly (A) Tailing

1. Add following reagents to same tube (s): 

36 µL MBG water

20 µL 5X E-PAP buffer

10 µL 25 mM MnCl2

10 µL ATP solution

4 µL E-PAP Enzyme

2. Incubate at 37ºC for 1 hour. 

3. After incubation, immediately add 10 µL ammonium acetate stop solution (same kit in Freezer B) to RNA tube(s) and mix by pipetting. 

**freeze for temporary storage

***GO NANODROP REMAINING LIN DNA 

D. Phenol:Chloroform Cleanup of RNA

1. Add 110 µL of acidic PCl (pH 4.5, Fridge B) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

2. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 g for 1 minutes.

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of isopropanol (Fridge A) and place in -20°C freezer (any normal freezer) for 30 minutes.

4. Centrifuge the tubes for 15 minutes at 13,000 rpm and 4°C. Use the centrifuge in the cold room on the 3rd floor.

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.

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

8. Re-suspend the dry RNA in 15 µL of MBG water (part of the same RNA kit, Freezer B).

Please put the RNA tube in an appropriate box in the -80 freezer (2nd floor).

Placed in CFTR RNA Box

1. 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.

2. Add reagents: 

24 µL MBG water

20 µL DNA (41.9 µg DNA)

5 µL 10X FD buffer (enzyme rack, FreezerA)

1 µL FD Nhe1 restriction enzyme **(incubate overnight for Nhe1, but for FD Nhe1 (FastDigest), do 2 hours)

3. 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 for 2 hours at 37°C to ensure complete digestion. Move to temporary rack in freezer A.

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). 

5. 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. 

Leaving to sit overnight in sodium acetate; resuming 07/21/2022

6. 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. 

7. 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. 07/21/2022

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

9. Re-suspend the dried DNA in 10 µL of MBG water (Fridge A).

RNA Synthesis: In Vitro Transcription

NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated. 

A. mMESSAGE mMACHINE T7 Ultra Kit (Freezer B)

1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Add the reagents in the following order and incubate at 37°C for 2 hours. Add a tally mark on the box.

mMessage mMACHINE T7 Ultra Kit

 10 µL T7 2X NTP/ARCA

 2 µL 10X T7 Reaction Buffer

 6 µL Linear DNA template

 2 µL T7 Enzyme Mix

Incubate for 2 hours at 37ºC

**Can freeze for temporary storage 

B. Turbo DNAse (same kit in freezer B) 

1. Add 1 µL Turbo DNAse to the RNA tube(s) and incubate at 37ºC for 15 min. 

C. Poly (A) Tailing

1. Add following reagents to same tube (s): 

36 µL MBG water

20 µL 5X E-PAP buffer

10 µL 25 mM MnCl2

10 µL ATP solution

4 µL E-PAP Enzyme

2. Incubate at 37ºC for 1 hour. 

3. After incubation, immediately add 10 µL ammonium acetate stop solution (same kit in Freezer B) to RNA tube(s) and mix by pipetting. 

**freeze for temporary storage

***GO NANODROP REMAINING LIN DNA 

D. Phenol:Chloroform Cleanup of RNA

1. Add 110 µL of acidic PCl (pH 4.5, Fridge B) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

2. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 g for 1 minutes.

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of isopropanol (Fridge A) and place in -20°C freezer (any normal freezer) for 30 minutes.

4. Centrifuge the tubes for 15 minutes at 13,000 rpm and 4°C. Use the centrifuge in the cold room on the 3rd floor.

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.

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

8. Re-suspend the dry RNA in 15 µL of MBG water (part of the same RNA kit, Freezer B).

Please put the RNA tube in an appropriate box in the -80 freezer (2nd floor).

Placed in CFTR RNA Box

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). 

5. 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. Leaving to sit overnight 08/03/2022

6. 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. 

7. 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. 08/04/22

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

9. Re-suspend the dried DNA in 10 µL of MBG water (Fridge A).

RNA Synthesis: In Vitro Transcription

NOTE: Take extra care in preparing RNA. Wear gloves and put on Eliminase as samples are easily contaminated. 

A. mMESSAGE mMACHINE T7 Ultra Kit (Freezer B)

1. Obtain a 1.5 mL tube and label with the name of the DNA plus RNA. Add the reagents in the following order and incubate at 37°C for 2 hours. Add a tally mark on the box.

mMessage mMACHINE T7 Ultra Kit

 10 µL T7 2X NTP/ARCA

 2 µL 10X T7 Reaction Buffer

 6 µL Linear DNA template

 2 µL T7 Enzyme Mix

Incubate for 2 hours at 37ºC

**Can freeze for temporary storage 

B. Turbo DNAse (same kit in freezer B) 

1. Add 1 µL Turbo DNAse to the RNA tube(s) and incubate at 37ºC for 15 min. 

C. Poly (A) Tailing

1. Add following reagents to same tube (s): 

36 µL MBG water

20 µL 5X E-PAP buffer

10 µL 25 mM MnCl2

10 µL ATP solution

4 µL E-PAP Enzyme

2. Incubate at 37ºC for 1 hour. 

3. After incubation, immediately add 10 µL ammonium acetate stop solution (same kit in Freezer B) to RNA tube(s) and mix by pipetting. 

**freeze for temporary storage

STOPPED HERE

Finished 08/14/2022

D. Phenol:Chloroform Cleanup of RNA

1. Add 110 µL of acidic PCl (pH 4.5, Fridge B) from the bottom layer. Vortex for 10 seconds and centrifuge at 13,000 rpm for 1 minute.

2. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of chloroform (Fridge A). Vortex for 10 seconds and centrifuge at 13,000 g for 1 minutes.

3. Transfer the top layer to a new, previously labeled 1.5 mL tube. Add 110 µL of isopropanol (Fridge A) and place in -20°C freezer (any normal freezer) for 30 minutes.

4. Centrifuge the tubes for 15 minutes at 13,000 rpm and 4°C. Use the centrifuge in the cold room on the 3rd floor.

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.

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

8. Re-suspend the dry RNA in 15 µL of MBG water (part of the same RNA kit, Freezer B).

Please put the RNA tube in an appropriate box in the -80 freezer (2nd floor).

A pellet maybe formed in one of them? I will run a gel on them to see what's going on

Placed in CFTR RNA Box

** GEL NOT RUN ON TWO 41.9 µg SAMPLES!!!!!!

1. Gather desired RNA samples and record the sample order and RNA volume added as explained in the DNA sample preparation protocol.

2. For each RNA sample, add: 

1 µL of RNA

2 µL of MBG water (Fridge A)

3 µL of 2X RNA Dye (Box 13, Freezer C)

3. The RNA ladder requires additional preparatory steps before it can be loaded.

a.       Combine 2 μL of ssRNA Ladder (Box 13, Freezer C) with 8 μL of 2X RNA Dye.

b.       Obtain a heating block and thermometer (both in Drawer B). Place the thermometer in the smallest hole and turn the ON/OFF switch to high. 

c.        Turn the high temperature control dial up to increase the temperature. Adjust the temperature according to the following settings: Incubate at 90°C for 2 minutes. 

d.       Immediately place it on ice for 2 minutes and load the entire volume into its corresponding well.

4. When the gel has set, it will have a certain glimmer to it and look solid. Lift the gel tray up and out of the tank. 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 in both sides of the tank and the gel is covered in TBE.

5. Spin the samples down before loading them. Set your pipette to ~6.5 µL to ensure complete transfer of the samples. Load samples according to the written order in the notebook. For the RNA ladder, load the entire volume into the well. Do not pierce the gel.

6. Slide the tank cover on ensuring that the negative prod is on the same side as the DNA. Turn the electrophoresis machine on by flipping the switch on the front. Run the gel for 90 minutes at 60 V.

RNA Ladder: RiboRuler (SM1823, Thermo Scientific)

If you load 4 µL of RiboRuler you get the following amounts in the bands. You may need to load 8 µL or more to see clear bands.