Standard Protocols

From 2006.igem.org

Latest revision as of 09:55, 21 August 2006

Resuspension of Dry DNA

1. Puncture a hole through the foil with a pipette tip
2. Add 15 ul of distilled water

Preparation of Liquid Cultures

1. Add 50 ml LB to 2 (or more) 250 ml conical flasks
2. Add 50 microlitres of ampicillin to each flask
3. Sterilise culture loop in a bunsen burner
4. Collect cells from the sample plate using the sterile culture loop
5. Innoculate the flasks with the cells from the loop
6. Sterilise the loop and repeat as necessary
7. Shake overnight at 37°C

Transformation into Competent Cells

1. Take 1ul of suspended DNA and add to an aliquot of competent E-Coli cells (taken from the freezer at -80°C)
2. Mix gently and leave in ice bucket for 10 to 30 minutes
3. Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
4. Return to ice for a short period of time (approximately 3 minutes)
5. Add 1 ml of LB liquid media
6. Shake for about an hour at 37°C
7. Plate on agar containing the correct antibiotic, put approx. 100ul of cells on one plate, and the remainder on the other after centrifuging and resuspending the pellet
8. Incubate at 37°C overnight
9. Remove from incubator and rest at room temperature for approximately an hour
10. Move to refrigerator

Restriction Digest

1. Decide on the final volume of the restriction digest (15ul for a test, 50ul for a preperative digestion)
2. 10% of the final volume should be buffer, less than 10% enzymes, more than 10% plasmid DNA and the final volume made up with water
3. Choose the correct enzymes to cut the plasmid with - EcoRI and PstI for a test, EcoRI and XbaI to open a plasmid vector or EcoRI and SbaI to cut out an insert to put in an opened vector
4. Allow the buffer to defrost, then vortex and centrifuge
5. If more than two samples are to be digested create a master mix by multiplying volume of buffer, enzyme and water by 10% more than the number of samples.
6. Put all reaction components together, then incubate at 37 degrees for two hours for a test, three hours for a preparative digestion

Test Gels

1. Make a 1%, or for smaller fragments 1.5% agarose gel (40ml and 0.4g of agarose for the small tank, 80ml and 0.8g agarose for the large tank)
2. Add the agarose to TAE buffer, then weigh and cook in the microwave at 70% power until particles are no longer visible
3. Weigh again, and add water to replace any liquid lost during microwaving
4. Cool, but don't set, and add ethidium bromide
5. Pour gel into tray sealed with tape at the ends, with appropriate combs in place
6. Wait to set
7. Add loading buffer to the samples for testing
8. Load wells with samples, and one well with DNA size markers
9. Stop gel after approx. 25 minutes, and view under UV light

Measurement of pH Change of lactose solution using E-Coli with and without lacZ

1. Prepare a saturated overnight culture of E-Coli with and without lacZ
2. Transfer 5 ml of culture to each of two 250 ml conical flasks (one with lacZ, one without) containing 50 ml of sterile LB medium
3. Add 50 microlitres of ampicillin to each flask
4. Incubate at 37°C with shaking for 30 to 60 minutes for cells to begin active growth.
5. Measure and record the OD in each flask
6. To each flask add 2.5 ml 20% w/v lactose and 50 microlitres of 90 mg/ml IPTG
7. Aseptically measure the initial pH of each flask.
8. Return the flasks to the 37 C shaker. Retest the pH after 15, 30, 45, 60 minutes, and then at further intervals depending on the results of these assays.

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