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From 2011.igem.org

Contents 1 E. coli Calcium Chloride competent cell protocol 2 REHYDRATION 3 TRANSFORMATION 4 Glycerol stock 5 Miniprep Plasmid DNA Isolation

E. coli Calcium Chloride competent cell protocol

• Materials

Equipment

• Centrifuge
• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• 0.1M CaCl2
• Glycerol

1. Inoculate a single colony (diameter: 2-3mm) into 100mL Lb in a falcon tube. Shake @ 37°C for 3hrs, 150-200rpm

a.From an ON culture (single colony into 5 mL LB in 50 mL falcon 37°C, 250 rpm), inoculate 1 mL into 100 mL LB (0.25 mL from ON culture into 25 mL LB in 50 mL falcon)

1. When the O.D. 600=0.35 put the cells on new tubes on ice for 10-15 mins (keep cold form now on).
2. Collect the cells by centrifugation 2700g (4100rpm) for 3 min at 4ºC
3. Decant supernatant (be careful not to dump out pellet, and drain tube on paper towel).
4. Gently resuspend on 10 mL cold 0.1M CaCl2 (cells are susceptible to mechanical disruption, so treat them nicely).
5. Incubate on ice for 15 min
6. Repeat 3 and 4.
7. Gently resuspend pellet on 2mL cold 0.1MCaCl2/15%Glycerol (2ml 0.1MCaCl2/50ml bacterial culture)
8. Dispense in microtubes. Freeze in -80°C.

REHYDRATION

• Materials

Equipment

• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• Distilled water

Biological agents

• Resuspended DNA. DNA kit plate

To use the DNA in the Distribution Kit you may follow these instructions:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells. But it is important to take care of the plate orientation after punching the foil.
2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 2µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.

TRANSFORMATION

• Materials

Equipment

• Incubator
• Water bath
• Petri dishes (with LB agar and appropriate antibiotic/ two per a transformation)
• Pipette tips
• Micropipettes
• Sterile tubes
• Sterile loops
• Racks

Reagents

• LB agar

• LB broth
• Distilled water
• Antibiotics (according to each BioBrick)

Biological agents

• Resuspended DNA
• Competent cells

1. Start thawing the competent cells on crushed ice.
2. Add 100 µL of thawed competent cells and then 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
3. Incubate the cells on ice for 30 minutes.
4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 2min. A water bath improves heat transfer to the cells.
5. Incubate the cells on ice for 5 minutes.
6. Add 200 μl of LB broth (make sure that the broth does not contain antibiotics and is not contaminated)
7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
9. Incubate the plate at 37ºC for 16 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.
10. Pick a single colony and inoculate broth (with the correct antibiotic) and grow for 16 hours.
11. Use the resulting culture to miniprep.

Glycerol stock

• Materials

Equipment

• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• 60ml glycerol: 4ml CaCl2: 36 ml de H2O
• 1ml glycerol/ 1 ml bacteria

1. Add 1 ml of 60% glycerol in H2O to a falcon tube.
2. Add 1 ml sample from the culture of bacteria to be stored.
3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.

a. Alternatively, pipet to mix.

1. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
2. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
3. Store in a freezer box in a -80ºC freezer. Remember to record where the vial is stored for fast retrieval later.

Miniprep Plasmid DNA Isolation

• Materials

Equipment

• Centrifuge
• Pipette tips
• Micropipettes
• Sterile tubes
• Racks

Reagents

• Solutions:

• Solution 1:

• 1.8g→ 50 mM glucose
• 0.6057g→ 25 mM Tris-HCl pH 8.0:
• 0.744g→ 10 mM EDTA pH 8.0:
• Add H2O to 200 ml.

• Solution 2:

• 2g→ 1% SDS. Add H2O to 198 ml.
• Add 1.6g→ 0.2 N NaOH
• Add H2O to 200 ml.

• Solution 3:

• 98.14g→ 5 M Potassium Acetate
• Add glacial acetic acid to 200 ml.

• TE:

• 0.2428g → 10 mM Tris-HCl pH 8.0
• 0.744g→ 1 mM EDTA
• Add H2O to 200 ml.

• Optional: RNAse can be added to TE at final concentration of 20 µg/ml.

Biological agents

Bacterial culture grown

1. Fill a centrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in centrifuge for 1 minute, and make sure tubes are balanced in centrifuge. Dump supernatant and drain tube briefly on paper towel.
2. Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel. If necessary, repeat this step (depending on the amount of pellet)
3. Add 2 ml ice-cold Solution 1 (GTE) to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

a.Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH (8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.

1. Add 4 ml Solution 2 (NAOH + SDS 1%), cap tubes and invert five times gently. Let tubes sit at room temperature for 5 minutes.
2. Solution 2 contains NaOH and SDS (a detergent). The alkaline mixtures ruptures the cells, and the detergent breaks apart the lipid membrane and solubilizes cellular proteins. NaOH also denatures the DNA into single strands.
3. Add 3 ml ice-cold Solution 3 (Potassium acetate), cap tubes and invert five times gently. Incubate tubes on ice for 10 minutes.

a.Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E. coli chromosomal DNA, a partially renatured tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in solution.

1. Centrifuge tubes for 5 minutes. Transfer supernatant to fresh centrifuge tube using clean disposable transfer pipet. Try to avoid taking any white precipitate during the transfer. It is okay to leave a little supernatant behind to avoid accidentally taking the precipitate.
2. This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.
3. Fill remainder of centrifuge tube with isopropanol. Let tube sit at room temperature for 2 minutes.
4. Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.
5. Centrifuge tubes for 10 minutes (13.4krpm max). A milky pellet should be at the bottom of the tube. Pour off supernatant without dumping out the pellet. Drain tube on paper towel.
6. This fractionation step further purifies the plasmid DNA from contaminants. This is also a good place to stop if class time is running out. Cap tubes and store in freezer until next class period.
7. Add 10 ml of ice-cold absolute ethanol. Cap tube and mix by inverting several times. Spin tubes for 1 minute. Pour off supernatant (be careful not to dump out pellet) and drain tube on paper towel.
8. Ethanol helps to remove the remaining salts and SDS from the preparation.
9. Allow tube to dry for ~5 minutes. Add 500 ul TE to tube. If needed, centrifuge tube briefly to pool TE at bottom of tube. DNA is ready for use and can be stored indefinitely in the freezer.