Team:UANL Mty-Mexico/Notebook/Protocols

1. Inoculate a single colony into 5 mL of LB media without any antibiotics and grow overnight at 37 °C with vigorous shaking.
2. Inoculate 1 mL of the desired strain into 100 mL of fresh LB, use a 500 mL flask.
3. Incubate at 37 °C with vigorous shaking until 0.3-0.4 OD₆₀₀
4. Put the flask on ice. Pre-chill 50 mL centrifuge tubes and the centrifuge itself at 4°C.
5. Centrifuge 50 mL of the culture at 8,000 rpm for 5 minutes at 4 °C.
6. Remove the supernatant and add 10 mL of cold CaCl₂ 0.1 M. Vortex until the pellet is resuspended. 
7. Incubate on ice for 30 minutes, shake the tube once in a while.
8. Centrifuge at 8,000 rpm for 5 minutes at 4°C. Remove the supernatant and add 2 mL of CaCl₂ 0.1 M. Resuspend carefully using a micropipette. Keep always on ice.
9. Mix the two preparations in a tube and store on ice, or use for transformation.

Note: The competent cells can be stored on ice up to two weeks.     

1. Add 50 µL of Ca⁺² competent cells to a pre-chilled centrifuge tube. Keep always on ice until step 4.
2. Add plasmid DNA (100 ng) or ligation (up to 5 µL) depending on DNA concentration.
3. Use 1µL of a 1 ng/µL DNA sample as positive test in a separate tube. It is recommended to use a DNA-free negative test tube as well.
4. Chill the tube on ice for 20-30 minutes.
5. Expose the reaction mixture to a 42ºC 1 minute heat-shock.
6. Put the tube on ice for 2 minutes.
7. Add 200 µL of antibiotic-free LB media.
8. Incubate at 37ºC for 20-30 minutes.
9. Spread the appropriate quantity of cells (50-200 µL) on selective LB agar plates.
10. Incubate overnight at 37º C.
11. The positive plate must have around 1,000 colonies as an optimal (1X10⁶ transformants per µg supercoiled DNA).

Notes:

Until heat-shock, handle the tubes from the upper part to avoid warming the cells. Low temperature is critical for successful transformation.

Avoid transforming with more than 5 µL of ligation mixture, as ligation buffer may reduce transformation efficiency.

1. Inoculate a single colony of E. coli in 5 mL of LB media. Grow overnight or for 5 hours at 37°C with shaking at 250rpm.
2. Inoculate 2.5 mL of the previous culture in 200 mL of LB media in a 2 L flask. Grow at 37 °C shaking at 300 rpm until the culture reaches an OD of 0.5-0.7. 
3. Chill the cells on ice for 10-15 minutes and then transfer the cells into a pre-chilled centrifuge bottle.
4. Centrifuge at 4,200 rpm for 10 minutes at 2 °C (Beckman J-6M).
5. Remove the supernatant and resuspend the pellet in 5 mL of cold water. Add 200 mL of cold water and mix well. Centrifuge at 4,200 rpm for 10 minutes at 2 °C.
6. Remove the supernatant and resuspend the pellet by shaking gently in the remaining liquid volume. 
7. Add 200 mL of cold water, mix well and centrifuge at 4,200 rpm for 20 minutes at 2°C.
8. Add 20 mL of 10% cold glycerol and mix well. Centrifuge at 4,200 rpm for 20 minutes at 2 °C.
9. Add 10 mL of 10% cold glycerol to each tube. Resuspend and gather all the content of the tubes in a single tube, centrifuge and remove the supernatant.
10. Estimate the pellet volume and add an equal volume of 10% cold glycerol. Resuspend the cells.
11. Divide the final volume into pre chilled tubes (100 μl) and store at -80 °C.

Note: Pre-chill all the materials that will be in contact with the cells.

1. Take a tube with 50 µL of electrocompetent E. coli cells, thaw on ice.
2. Add a volume containing 100 ng of DNA.
3. Carefully transfer the cell/DNA mix into a pre-chilled electroporation cuvette. Make sure to deposit the cells at the bottom and not to introduce any air bubbles.
4. Electroporate under the following conditions:
5. Immediately add 250 µl of SOC media to the cuvette.
6. Incubate with vigorous shaking (250 rpm) at 37 °C for 1 hour.
7. Add 750 µL of LB media and mix by pipetting up and down.
8. Spread 200 µL of cells onto a selective LB agar plate. 

Note: All must be performed on ice. Electroporation cuvettes are previously chilled on ice. DNA and bacteria must be thawed on ice too. 

1. Pour 1.5 ml of the culture in a 1.5 ml microcentrifuge tube and centrifuge at 14,000 rpm for 30 seconds. Remove carefully the supernatant.
2. Add 200 µl of Solution I. Resuspend the pellet by using vortex briefly or by pipetting up and down. Incubate at room temperature for 5 minutes. 
3. Add 200 µl of Solution II and mix gently by inverting and rotating the tube several times. Do not vortex. Incubate at room temperature for 5 minutes.
4. Add 200 µl of Solution III and mix gently by inverting and rotating the tube several times. Incubate the tube on ice for 5 minutes.
5. Centrifuge at 14,000 rpm for 5 minutes.
6. Transfer the supernatant to a fresh tube containing 1 mL of 100% ethanol.
7. Incubate at -20 ºC for 10 minutes. (Max. 2 h)
8. Centrifuge at 14,000 rpm for 10 minutes. Remove the supernatant.
9. Add 200 µl of 70% ethanol and vortex gently for 10 seconds.
10. Centrifuge at 14,000 rpm for 5 minutes. Remove the supernatant by pipetting. Aspirate off any residual supernatant.
11. Dry at 37 ºC for 5 minutes.
12. Add 20 µl of H₂O + 20 µg/mL of RNase. Resuspend by using vortex briefly.
13. Run an agarose gel (0.8%) or store at 4 ºC.                       

Notes:

When preparing Solution II, first add a little bidistilled water, then add NaOH and dissolve carefully SDS. Finally, bring the final volume up to 200 ml with bidistilled water. 
When preparing Solution III, first add 70 ml of H₂O and then the potassium acetate. Once it has been dissolved add the acetic acid and finally bring the final volume up to 100 ml with bidistilled water.

To be loaded:

DNA molecular size marker (λ PstI): 2 - 3 μL

Plasmid DNA: 2 - 3 μL

Enzyme restrictions: 10 μL

PCR products: 5 μL

Procedure:

1. Prepare an agarose gel of the desired concentration (see Agarose gels section).
2. Add the necessary SB 1X buffer into the electrophoresis tank to cover the gel.
3. Load the first well with marker, and then load the DNA samples mixed with loading buffer into the wells.
4. Plug in the anode and cathode cables so that the DNA samples can move through the gel toward the anode.
5. Run the electrophoresis at 200 volts.
6. Wait approximately 20-30 minutes or until the bromophenol blue reaches the end of the gel and stop the electrophoresis.

Note: DNA moves toward the positive electric field (anode, usually red cable) due to the negative charges.

Agarose gel concentration needed for supercoiled DNA

and mini preparation of plasmid DNA 0.8%

For digestion reaction fragments over 1,000 bp 0.8%

For digestion reaction fragments below 500 bp 1.5%

DNA size marker (λ + PstI)

Use 2 or 3 μL per gel.

Note: Is not needed when running supercoiled DNA samples, like plasmid DNA.

iMAGEN:LAMBDA

SB buffer:

It is prepared from a SB 20X Stock using bidistilled water. Use 50 ml of SB 20X buffer when preparing a liter of SB 1X. 

It can be used even at 200 volts in an electrophoresis. 

Notes:

At this condition the electrophoresis will end in approximately 20 minutes.

In case of running agarose gels with digestion products, verify the size of the resulting fragments so that the smallest fragments do not get out of the gel.

1. Dilute the stock to 20 μg/mL in a special container with the gel buffer.
2. Put the gel into the container.
3. Let it stain for 3 - 5 minutes.
4. Take the gel out of the container and soak the stained gel in water for 5 minutes or more to clear background ethidium bromide from the gel.
5. View the gel under a UV light source or on a UV transilluminator.

Note: If you want to use ethidium bromide, confine its use to a small area of your laboratory. Wear gloves when staining, handle stained gels, and dispose of any waste. 

Mix:

• 49.6 μL phage λ DNA (504 ng/μL)
• 2.5 μL PstI
• 6 μL Buffer II
• Total: 58.1 μL

Protocol:

1. Mix the ingredients listed above
2. Incubate at 37ºC/45 min
3. Again add 2.5 μL PstI
4. Incubate 37ºC/45 min
5. Add the following:

• 0.75 μL EDTA (0.5 M)
• 12.8 μL Loading buffer 6X (7.68 μL of 10X buffer)
• 1.45 μL H2O (6.57 μL of buffer 10X buffer)
• Total: 15 μL

Check on agarose gel.




Notes:

Final concentration: 0.33 μgr/μL, 5.9 ng/kb

Final Volume: 75.6 μL




Restriction enzyme digestion of DNA

Mix for 1 reaction, final volume (20 µL)




Add the following to a microcentrifuge tube:




DNA	2-4 µg
Buffer 10x	2.0 µL
Enzyme (10 U/µL)	0.3 µL
H2O	Until 20.0 µL










Incubate the mixture at 37 °C (it depend of each enzyme specification) for 1-1.5 hours.




Note: Prepare a mix when possible to minimize enzyme handling. 




PCR

Composition of the PCR mix

Final volumen (25 µL)




DNA template	Total 100 ng (In 25 μl)
Buffer 10x	2.5 μL
Mg++ 50 mM	0.75 μL
dNTPs 20 mM	0.25 μL
Primer Fwd 100 ng/μL	0.50 μL
Primer Rv 100 ng/μL	0.50 μL
Taq Pol 5 U/μL	0.25 μL
H2O	To bring the volume up to 25 μL







Procedure:

1. Take a sterile PCR tube and first add the H₂O.
2. Then, add the other components, except the enzyme and the DNA.
3. Prepare the premix like you have calculated. As last, add the enzyme mix by vortex and dispense in the individual PCR tubes, add the respective DNA template.
4. Gently mix the reacction.
5. Centrifuge it for 10 seconds.
6. Place the samples in the thermocycler and start your PCR program.




Notes:

Put on gloves before you take the PCR mix components out of the freezer box.

DNA is the last to be added because it forms complexes with Mg⁺⁺ and that inhibits the reaction.

If preparing several reactions using the same enzyme, first prepare a mega-mix containing all the components for all the reactions except DNA template, and divide the mix over the PCR tubes, finally add the respective DNA template.




Antibiotics

Antibiotic	Final concentration	Stock concentration	µL per mL
Spectinomycin       (Sp)	100 µg/mL	20 µg/µL	5
Ampicillin              (Amp)	50 µg/mL	10 µg/µL	1
Kanamycin            (Kan)	50 µg/mL	50 µg/µL	1
Chloramphenicol   (Cm)	34 µg/mL	34 µg/µL	1
Tetracycline           (Tet)	10 µg/mL	5 µg/ µL	2







Notes: 

• Always check the concentration of Stock solution in order  to calculate the needed volume of antibiotic.
• In case of unknown concentration, use the concentration indicated in the table.
• If there is no time to do the math, add 1 µL per mL of the indicated antibiotic.
• When using several antibiotics at the same time is recommended to use the half of the concentration for each antibiotic.