Some common techniques used with our plasmids are listed below. For a more complete description of these and other methods please refer the following publication:
Sambrook, Joseph. and Russell, David W. and Cold Spring Harbor Laboratory. Molecular cloning : a laboratory manual, Joseph Sambrook, David W. Russell Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. : 2001Transformation (Chemical)
Transformation protocols vary based on the manufacturer and the cell line. For best results we recommend you follow a protocol optimized for the particular cells you are using. Below is a general protocol applicable for many commercial E. coli cell lines such as DH5α or XL1 Blue.
Place frozen competent cells and pre-labeled tubes on ice
- Pre-warm the water bath to 42°C
- Once cells are thawed on ice, gently aliquot 25 µL of XL1 Blue cells (Stratagene) to each of the pre-chilled tubes. Keep all the tubes on ice
- Add 2 µL of the diluted plasmid (0.4ng) to the cells and gently swirl the cells with pipette tip
- Incubate on ice for 30 minutes
- Heat shock tubes for 45 seconds in 42°C hot bath
- Return tubes to ice for 2 minutes
- Add 250 µL of SOC media (recipe below) to each tube and place tubes in a 37°C shaking incubator for 1 hour
- Pre-warm agar plates to 37°C in incubator
- Spread 125 µL of cells on agar plates containing the appropriate antibiotic
- After spreading cells, let plates sit for approximately 10 minutes at room temperature. Then place the plates inverted in 37°C incubator for 12 – 24 hour
Inoculation and plasmid isolation
- For a large number of colonies, use a 2.2 mL deep well plate and add 1.6 mL of LB Broth with the appropriate antibiotic. For a small number of colonies, use a 14 mL round bottom culture tube and add 2 mL of LB Broth with the appropriate antibiotic
- Touch the colony with a toothpick or pipette tip and place it in the broth
- Cover with a gas permeable lid and place in a 37°C shaking incubator for 12 – 20 hours.
- Plasmid DNA can be purified from broth cultures by a variety of commercial kits or by the alkaline lysis method listed in the Molecular Cloning manual by Sanbrook et al. listed above
Restriction enzyme Digestions
The standard in-house digestion protocol is below:
Incubate for 1 hour at the temperature recommended by the enzyme manufacturer. Take 2-5 µL of the digested sample, add loading dye, and run on a gel to verify that the digestion took place. In separate lanes, also run a ladder to verify the size of the product and a sample of undigested product as a control.
*The length of running time for the gel will depend on the size of the digested insert. Smaller inserts require shorter run times or the product will run off the gel.
*The enzyme should not be more than 1/10th of total reaction
| DNA | 400ng |
| Buffer | 5µL |
| Water | XµL |
| Enzyme(s) | 1µL each |
| Total Volume | 50µL |