Team:Cairo Egypt/Experiments

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Experiments and Protocols

General Preparation

Gel Preparation

1. Prepare 50 mL of 0.7% agarose gel. Add 0.35 g of agarose to 50 mL of TAE buffer and boil in microwave until all agarose is melted. Stop microwave every 20 to 30 seconds and swirl the solution to help the agarose dissolve. Required about 2 minutes for all of the agarose to dissolve. a. 50mL 1X TAE buffer i. 10 mL of 50X TAE ii. 490 mL of DI water
2. Let agarose gel solution cool to about 55C (hot but not burning to the touch). This will take only a few minutes. Take gel solution to the Gulari lab and add 10 uL of ethidium bromide (10 mg/mL). Swirl solution to mix for at least 30 seconds
3. Get about a 9 cm gel cast and put barriers at each end (make sure they are snug). Pour gel solution into cast, try not to create bubbles/foam.
4. Use pipette tip to pop any bubbles. If bubbles will not pop, use tip to move the to the sides of the gel.
5. Put comp into gel to create wells. Use the 13x comb. Put the comb near the beginning of the gel, second notch from the rubber stop. Leave gel on bench 20-25 minutes to solidify.

Preparation of Antibiotics

1. Ampicillin 100 µg/mL
2. Tetracycline 10 µg/mL
3. Chloramphenicol 25 µg/mL
4. Kanamycin 50 µg/mL

Preparation of LB media

1. Dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.
2. Adjust the pH of the medium to 7.0 using 1N NaOH and bring volume up to 1 liter.
3. Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 55°C, and add antibiotic if needed (50µg / mL of Amp or Kan).
4. Store at room temperature or +4°C.

Preparation of LB-Agar

1. Prepare LB medium as above, but add 15 g/L agar before autoclaving.
2. After autoclaving, cool to approx. 55°C, add antibiotic (if needed), and pour into petridishes.
3. Let harden, then invert and store at +4°C in the dark

Protocol To make 500mL of LB agar (makes about 25 LB agar plates):

Weigh out the following into a 1L Erlenmeyer flask:

• 5g NaCl
• 5g Tryptone
• 2.5g Yeast Extract
• 7.5g Agar
• add dH2O to 500mL

Preparation of reagents for manual plasmid mini prep

P1: 1 molar Tris PH 8 5ml, .5 molar EDTA 2ml, glucose, Distilled water up to 100ul

P2: .2 molar NAOH 1% SDS

P3: 5 molar sodium acetate (adjusted with glacial acetic acid ) or 2.55 molar potassium acetate.

Plasmid Mini prep by alkaline lysis

1. Take 15 ml over night culture, centrifuge at max speed 10 minutes
2. Remove the supernatant and re-suspend the Pellet in 2 ml resuspension buffer
3. Add 4 ml lysis buffer, mix by invesion
4. Incubate 5 mins at RT, add 3ml neutralization buffer, incubate 15 mins on ice.
5. Centrifuge at max speed for 10 mins "Cooling centrifuge 1000g"
6. Take about 8 ml supernatant and add 0.6vol isopropanol
7. Centrifuge at Max speed for 10 mins then remove the supernatant
8. Wash the pellet using 70% ethanol, discard the ethanol and then air dry
9. Add 200ul TE buffer to the pellet "Resuspend and transfer to new epp"
10. Add another 200ul TE buffer to the new eppendorf
11. Add 10ul RNAse then incubate 15 mins at 37C
12. Add equal volume of Phenol chloroform , vortex, centrifuge at max speed 2 mins
13. Transfer the upper layer to new epp then add equal volume of chloroform, vortex, centrifuge at max speed 2 mins
14. Transfer the upper layer to new eppendorf the add 1/10 vol 3M Na.acetate then add 2.5vol absolute ethanol.
15. Centrifuge 15 mins at 4C
16. Discard supernatant, Wash the pellet using 70% ethanol, remove the ethanol then spin down
17. Re-suspend the pellet in TE buffer
18. Measure the concentration and purity

Making a glycerol stock :

1. Pick a single colony of the clone off a plate and grow an overnight in the appropriate selectable liquid medium (3-5ml).
2. Add 0.5 ml of 40% glycerol in H2O to a cryogenic vial.
3. Add 0.5 ml sample from the culture of bacteria to be stored.
4. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. Alternatively, pipet to mix.
5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
6. Freeze glycerol stock in liquid nitrogen and store in a -80C freezer. This will also be a good time to record the strain information and record the location

Transformation from the registry

(from the competition protocols)

gel purification for PCR products "thermo scientific kit"

Before Starting. Add ethanol to Wash Buffer (W1) according to the label on the bottle. Equilibrate a water bath or heat block to 50°C.

1. Excise. Use a clean, sharp razor blade to excise a minimal area of gel containing the DNA fragment of interest.
2. Using a scale sensitive to 0.001 g, weigh the gel slice containing the DNA fragment.
3. Add Gel solubility Buffer (L3) to the excised gel in a tube as indicated in the table. Incubate the tube at 50°C for 10 minutes (or longer for large gel slices and high concentration gels), and invert the tube every 3 minutes. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes. (Optional) Add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
4. Pipet the dissolved gel piece onto a column inside a Wash Tube. Centrifuge column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Note: The column reservoir capacity is 850 µL. Use 1 column per 400 mg of agarose gel.
5. Add 500 µL Wash Buffer (W1) containing ethanol to the column. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes. Discard the flow-through.
6. Elute. Place the column into a Recovery Tube. Add 50 µL Elution Buffer (E1) to the column. Incubate the tube for 1 minute at room temperature. Centrifuge the tube at >12,000 × g for 1 minute. 7. Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.

• Restriction digestion reaction (Takara protocols)
• Restriction digestion (thermo scientific fast digest)
• Rapid ligation kit (Thermo scientific)

Ligation Protocols:

Ligation of insert DNA into plasmid vector DNA Thoroughly mix the 5X Rapid Ligation buffer prior to use.

1. Add the following to a microcentrifuge tube:
   • Linearized vector DNA 10-100 ng
   • Insert DNA (at 3:1 molar excess over vector) variable
   • 5X Rapid Ligation Buffer
   • 4 μl T4 DNA Ligase
   • 5 u/μl 1 μl Water
   • nuclease-free to 20 μl
   • Total volume 20 μl
2. Vortex and spin briefly to collect drops.
3. Incubate the mixture at 22°C for 5 min.
4. Use 2-5 μl of the ligation mixture for transformation. The reaction mixture can be stored at 0-4°C until used for transformation. Prior to electroporation, chloroform extract the ligation mixture and use 1 μl for the electroporation reaction.

Restriction digestion using (ECORi 1)

Material

• Ice and bucket/container
• (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
• BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
• dH2O
• NEB Buffer 2
• BSA
• Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
• Thermal cycler

Notes

• You should keep all materials on ice.
• At iGEM HQ we use restriction enzymes from New England Bio labs

Single Reaction

1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
2. Add 2.5ul of NEBuffer 2.
3. Add 0.5ul of BSA.
4. Add 0.5ul of EcoRI.
5. Add 0.5ul of PstI.
6. There should be a total volume of 20ul. Mix well and spin down briefly.
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated
8. Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.

Preparation of competent cells "all steps on ice & between 2 flames"

1. Take 1 ml from over night culture, then added to 50 ml of fresh media (LB)
2. Shake 2 hrs at 37C in shaking incubator (then calculate the OD600nm "must reach 0.3-0.5")
3. Centrifuge to harvest the cells (3000 rpm for 10 mins)
4. Remove the supernatant, add 20 ml of 50mM cacl2
5. Leave on ice for 20 mins
6. Spin down for 10 mins at 8000 rpm
7. Re-suspend the pellet in 1 ml 50mM ice cold cacl2
8. Incubate on ice for 1 hr
9. Add glycerol 1 ml (Stock 40%)