Standard Protocols
From 2006.igem.org
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| - | === Resuspension of | + | === Resuspension of Dry DNA === |
# Puncture a hole through the foil with a pipette tip | # Puncture a hole through the foil with a pipette tip | ||
# Add 15 ul of distilled water | # Add 15 ul of distilled water | ||
| + | |||
| + | === Preparation of Liquid Cultures === | ||
| + | |||
| + | # Add 50 ml LB to 2 (or more) 250 ml conical flasks | ||
| + | # Add 50 microlitres of ampicillin to each flask | ||
| + | # Sterilise culture loop in a bunsen burner | ||
| + | # Collect cells from the sample plate using the sterile culture loop | ||
| + | # Innoculate the flasks with the cells from the loop | ||
| + | # Sterilise the loop and repeat as necessary | ||
| + | # Shake overnight at 37°C | ||
=== Transformation into Competent Cells === | === Transformation into Competent Cells === | ||
| Line 9: | Line 19: | ||
# Mix gently and leave in ice bucket for 10 to 30 minutes | # Mix gently and leave in ice bucket for 10 to 30 minutes | ||
# Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes) | # Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes) | ||
| - | Return to ice for a | + | # Return to ice for a short period of time (approximately 3 minutes) |
| + | # Add 1 ml of LB liquid media | ||
| + | # Shake for about an hour at 37°C | ||
| + | # 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 | ||
| + | # Incubate at 37°C overnight | ||
| + | # Remove from incubator and rest at room temperature for approximately an hour | ||
| + | # Move to refrigerator | ||
| + | |||
| + | === Restriction Digest === | ||
| + | |||
| + | # Decide on the final volume of the restriction digest (15ul for a test, 50ul for a preperative digestion) | ||
| + | # 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 | ||
| + | # 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 | ||
| + | # Allow the buffer to defrost, then vortex and centrifuge | ||
| + | # 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. | ||
| + | # Put all reaction components together, then incubate at 37 degrees for two hours for a test, three hours for a preparative digestion | ||
| + | |||
| + | === Test Gels === | ||
| + | |||
| + | # 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) | ||
| + | # Add the agarose to TAE buffer, then weigh and cook in the microwave at 70% power until particles are no longer visible | ||
| + | # Weigh again, and add water to replace any liquid lost during microwaving | ||
| + | # Cool, but don't set, and add ethidium bromide | ||
| + | # Pour gel into tray sealed with tape at the ends, with appropriate combs in place | ||
| + | # Wait to set | ||
| + | # Add loading buffer to the samples for testing | ||
| + | # Load wells with samples, and one well with DNA size markers | ||
| + | # 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 === | ||
| + | |||
| + | # Prepare a saturated overnight culture of E-Coli with and without lacZ | ||
| + | # 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 | ||
| + | # Add 50 microlitres of ampicillin to each flask | ||
| + | # Incubate at 37°C with shaking for 30 to 60 minutes for cells to begin active growth. | ||
| + | # Measure and record the OD in each flask | ||
| + | # To each flask add 2.5 ml 20% w/v lactose and 50 microlitres of 90 mg/ml IPTG | ||
| + | # Aseptically measure the initial pH of each flask. | ||
| + | # 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. | ||
| + | [http://2006.igem.org/University_of_Edinburgh_2006 Main page] | ||
| - | + | __NOTOC__ | |
| - | + | ||
| - | + | ||
Latest revision as of 09:55, 21 August 2006
Resuspension of Dry DNA
- Puncture a hole through the foil with a pipette tip
- Add 15 ul of distilled water
Preparation of Liquid Cultures
- Add 50 ml LB to 2 (or more) 250 ml conical flasks
- Add 50 microlitres of ampicillin to each flask
- Sterilise culture loop in a bunsen burner
- Collect cells from the sample plate using the sterile culture loop
- Innoculate the flasks with the cells from the loop
- Sterilise the loop and repeat as necessary
- Shake overnight at 37°C
Transformation into Competent Cells
- Take 1ul of suspended DNA and add to an aliquot of competent E-Coli cells (taken from the freezer at -80°C)
- Mix gently and leave in ice bucket for 10 to 30 minutes
- Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
- Return to ice for a short period of time (approximately 3 minutes)
- Add 1 ml of LB liquid media
- Shake for about an hour at 37°C
- 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
- Incubate at 37°C overnight
- Remove from incubator and rest at room temperature for approximately an hour
- Move to refrigerator
Restriction Digest
- Decide on the final volume of the restriction digest (15ul for a test, 50ul for a preperative digestion)
- 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
- 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
- Allow the buffer to defrost, then vortex and centrifuge
- 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.
- Put all reaction components together, then incubate at 37 degrees for two hours for a test, three hours for a preparative digestion
Test Gels
- 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)
- Add the agarose to TAE buffer, then weigh and cook in the microwave at 70% power until particles are no longer visible
- Weigh again, and add water to replace any liquid lost during microwaving
- Cool, but don't set, and add ethidium bromide
- Pour gel into tray sealed with tape at the ends, with appropriate combs in place
- Wait to set
- Add loading buffer to the samples for testing
- Load wells with samples, and one well with DNA size markers
- 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
- Prepare a saturated overnight culture of E-Coli with and without lacZ
- 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
- Add 50 microlitres of ampicillin to each flask
- Incubate at 37°C with shaking for 30 to 60 minutes for cells to begin active growth.
- Measure and record the OD in each flask
- To each flask add 2.5 ml 20% w/v lactose and 50 microlitres of 90 mg/ml IPTG
- Aseptically measure the initial pH of each flask.
- 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.
[http://2006.igem.org/University_of_Edinburgh_2006 Main page]
