EXPERIMENTS

We went through how we designed the project here . Here, we go over the specific protocols that we used throughout the course of the project

Protocols

Making Electrocompetent cells:

Prepare:

10% glycerol stock

Cold dH₂O

Bacteria

Escherichia coli

1. Grow bacteria until the OD should be around 0.4

2. Transfer the media w/ bacteria to a bottle

3. Centrifuge it at maximum speed for 25 minute

4. Pour the supernatant gently after finishing step 3

5. Add a tiny amount of water into the bottle and resuspense the bacteria

6. Transfer them to a Falcon tube

7. Add water until it reaches 500 mL

8. Centrifuge it for 7 minutes

9. Repeat 4-8

10. Wash them with 10% glycerol

11. Pour the supernatant

12. Resuspense them and check if it works with the electroporation machine

13. If it works, aliquot 20 uL to PCR tubes

14. If not, wash them again with 10% glycerol and then aliquot them

15. Store them in -80˚ Celcius

For Lactobacillus

1. Grow bacteria until the OD is around 0.4

2. Transfer them to two Falcon tubes

3. Centrifuge them at 4˚ Celcius for 7 minutes at maximum speed

4. Pour the supernatant gently

5. Fill the tubes with 0.5 M sucrose 10% glycerol until the volume reaches 50 mL

6. Repeat 3-5 twice

Electroporation

Escherichia coli

1. Use the brown cap cuvette

2. Mix 50 ng of DNA and 20 uL of E. coli

3. Transfer them to the cuvettes. Make sure it all goes through the bottom and there's no air bubble.

4. Electroporate (The timing should be > 5ms)

5. Add recovery buffer

Lactobacillus

1. Use the blue cap cuvette (The wider one)

2. Mix 2 ug of DNA and 60 uL of bacteria

3. Transfer them to the cuvettes

4. Electroporate (The timing should be >9 ms)

5. Add 1 mL MLS instead of recovery buffer

MiniPrep

1. Pellet 1-5 mL bacterial overnight culture by centrifugation at >8000rpm for 3 minutes at room temperature (15-25 C)

2. Resuspend pelleted bacterial cells in 250 uL Buffer P1 and transfer to a micro centrifuge tube

3. Add 250 uL of Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 minutes if using lyseBlue reagent, the solution will turn blue

4. Add 350 uL of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless

5. Centrifuge for 10 minutes at 13000 rpm in a table-top micro centrifuge

6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 seconds and discard the flow through or apply vacuum to the manifold to draw the solution through the QIAprep spin column and switch off the vacuum source

7. Recommended to wash the QIAprep sin column by adding 500 uL of Buffer PB and centrifuge that for 30-60 seconds discard the flow and or vacuum to the manifold to draw the solution through the QIAPrep spin column and switch off the vacuum source

8. Wash the QIAprep spin column by adding 750 uL of Buffer PE centrifuge that for 30-60 seconds and discard the flow

9. Centrifuge for a minute

10. Place the QIAprep column in a clean 1.5 ml micro centrifuge tube to elute DNA add 50 uL of Buffer EB or water to the center of the QIAprep spin column, let it stand for a minute and then centrifuge it for a minute

Dot Blotting

1. Measure the OD of the cell culture

2. Make buffers if necessary

a. So get falcon tubes and label them wash, lysis, elution

Lysis	10 mL NaH2PO4	75 uL NaCl	5 uL imulsion
Wash	10 mL NaH2PO4	75 uL NaCl	5 uL imulsion
Elusion	10 mL NaH2PO4	75 uL NaCl	5 uL imulsion

3. For the buffers when you finish adding the material then you fill them with water and mix it gently fill it up to 50 mL

4. Before this the culture tubes are placed in the centrifuge for 10 minutes to spin down the culture so that you be able to see the cells at the bottom (make sure to balance it out)

5. Then grab the spin tubes (the light blue ones) label these

6. Then get e.tubes and label them

7. In the falcon skinny purple tubes label them and remove the supernatant in the falcon tube and the supernatant remains in the culture tube

8. Add glass beads in the e.cubes and other dark beads that are found on Sonja bench

a. You should fill the e. tubes up to 0.1 mark

9. Add 200 uL of 20 mM tris HCl to the cell culture and then transfer that to e.tubes (these are the ones that contain the beads)

10. Add 600 uL of sodium phosphate to the new falcon tube that contains liquid supernatant

11. Add 4.5 uL of 2 M NaCl into these falcon tubes

12. Lysis buffer add 600 uL to the blue tubes

a. And centrifuge this for 2 minutes at 2000 rpm

13. Use the sonfication machine

a. Do this 3 times and have a 1 minute rest in between

14. Once you take out of the sonification machine place them in the centrifuge machine for 10 minutes at the speed of 14000 rpm

15. After transfer the liquid into new e.tubes and throw out the other part

16. In the blue spin tubes place 600 uL from the purple tube and centrifuge for 2 minutes at 2000 rpm

17. Throw out the liquid and place 600 uL again

a. You have to continue to do this step until you are done with the supernatant (so keep repeating this step)

18. Then add wash buffer 600 uL after done with previous step

a. Do this 3 times and centrifuge for 2 minutes

19. Then add 30 uL of elusion buffer this is done once

20. After the 2 minutes you take the liquid in the white e.tube and transfer to the blue tube again and place it back

a. Do this twice

21. Label the petri dishes

a. On the bottom side and on the side of the plate

22. Cut out the nitrocellulose membrane and label it on one side

a. So that you be able to determine which is the lysate side and the supernatant side

23. Then do the serial dilutions

a. You have to do 1 in 10, 1 in 100, and 1 in 1000

b. So in e. tubes you add 90 uL of water and then 10 uL of pure sample to the 1 in 10 then obtain 10 uL of that into 1 in 100 and then obtain 10 uL and place into 1 in 1000 tube

c. Do this for both lysate and the other sample (pure)

24. Then place the membrane on the petri dishes

a. Then begin dot blotting by inserting 5 uL of each sample including the serial dilutions, make sure to go down the column one side is the supernatant and the other is the lysate

b. Make sure to be neat about this (TRY your best on doing them in a straight line)

25. Then after all dots are placed dry it with the heat gun

a. By doing this you have to hold down the membrane with the tonsils and apply the heat gun on it for about a few seconds until the dot is completely dried off

i. Do this for all the petri dishes

26. Then add the buffer to it

a. Wash it 2 times for 10 minutes

27. After this add a blocking buffer

a. Wait for an hour

For the rest follow the protocol for dot blotting:

· In order to make the antibodies, they are located in the fridge it is the one with the one dot and the other is labeled

· You have to take 1.5 grams of the buffer that was used before, which it mentions on the sheet.

· Measure that amount and place into the falcon tube and then add TMS the buffer and shake it gently back and forth.

· After this add the antibody in the tube and label this will be used in the further steps

Team:Columbia NYC/Experiments

EXPERIMENTS

Protocols