Locate the Part

Use the Registry tools to locate the desired part. It is important to note the plasmid and antibiotic resistance(s) of the required part. See more information [http://partsregistry.org/Help:IGEM_08_DNA_distribution#Locating_a_Part_in_the_Distribution Here].

Paper Punches

Estimated time: 1 minute per spot

Materials needed:

1. Warm up 5 μl aliquots of TE buffer to 50ºC in 0.5 ml (PCR) Eppendorf tubes. Warm a water bath to 42 degrees.
2. Slide the cutting mat under the page in the binder containing the DNA of your part.
3. Using the punch tool, press down firmly on the spot you want to punch out while rotating the punch tool. The spot is large enough to allow several punches, so punch at the edge of the spot.
4. Eject the punched paper spot into the tube containing 5 µL of TE buffer by pressing down on the top part of the punch tool.
5. Be sure to clean the punch tool before punching out another spot, to prevent cross-contamination of parts.

Punch Tool Cleaning

Estimated time: 5 minutes per spot

Materials needed:

1. Punch a blank sheet of blotting paper (back of the notebook) and discard the punched paper. Pull the punch tool apart, so that the black rod is separated from the column.
2. Dip both the rod and the column briefly into a series of solutions of 10% bleach, distilled water, a second bath of distilled water, and a final bath of 95% ethanol. We have found that strong detergents tend to remain on the punch tool and can inhibit transformation of the DNA. Ethanol is preferred over isopropanol for the final cleaning bath, as it dries much faster.
3. Blot with a Kimwipe and allow to dry for 5 minutes. Note that the ethanol can wick up inside the column of the punch tool. It is important that the punch tool be completely dry before punching out another DNA spot, as the ethanol will affect the transformation efficiency.

Transformation

Estimated time: 3 hours (plus 12-14 hour incubation)

Materials needed:

1. Soak the spots in 5 µL of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice.
2. Chill labeled 2 ml conical bottom tubes on wet ice. Add 2 µL of DNA in TE and 50 µL of thawed TOP10 competent cells to the tubes. In our experience, these volumes have the best transformation efficiency. The 2 ml tubes allow better liquid movement during incubation. Extra eluted DNA may be held at least several weeks frozen or at refrigerator temperature.
3. Hold the DNA and competent cells on ice for 30 minutes. This improves transformation efficiency by a significant amount.
4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath is important to improve heat transfer to the cells.
5. Incubate the cells on ice for 2 minutes.
6. Add 200 μl of SOC broth (check that this broth is not turbid, which would indicate previous contamination and bacterial growth). This broth should contain no antibiotics.
7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. We have found that growth for 2 hours helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
8. Label an LB agar plate containing the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 250 µl of the incubated cell culture on the plate.
9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.