Revision as of 18:07, 27 July 2015

Transformation Protocol

Estimated time: 3 hours (plus 14-18 hour incubation)

Transformations are essential to using the DNA Distribution Kits: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies. However, transformations can also be one of the more fickle laboratory techniques.

At iGEM HQ, we run test transformations of the DNA Distribution Kit with the following protocol. We have found that it is the best protocol to use with Registry samples and ensures high efficiency transformations. This protocol may be particularly useful if you are finding that your transformations are not working, or yielding few colonies.

Important Reminders

• At iGEM HQ, we make our own stocks of NEB 10b competent cells. Competent cells purchased from vendors will have better efficiency.
• Make sure to test the competency of your cells. We provide a Competent Cell Test Kit to test the competency of your cells with different concentrations of DNA.
• Read through the entire protocol before starting!

Materials

• Resuspended DNA (Resuspend well with 10ul dH20, pipette up and down several times, let sit for a few minutes. Resuspension will be red, from cresol red dye.)
• 10pg/ul Control (pSB1C3 w/ BBa_J04450)
• Competent cells (50ul per transformation. We store competent cells in aliquots of 260ul for a total of 5 reactions)
• Ice (in ice bucket/container)
• 2ml tube (1 per a transformation. We recommend labelling tubes before getting started.)
• 42ºC water bath
• SOC media (check for contamination!)
• Petri dishes with LB agar and appropriate antibiotic (2 per transformation, for a 20ul and 200ul plating)
• sterile glass beads or spreader
• 37ºC incubator

Procedure

1. Thaw competent cells on ice. This may take 5-10min for a 260ul stock. Pre-chill empty 2ml tube(s) for each transformation.
2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
3. Add 1 - 2 µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
4. Add 1 µL of the 10pg/ul Control to your control transformation. Pipet up and down a few times, gently.
5. Close tubes and incubate the cells on ice for 30 minutes.
6. Place 2ml tubes in float, and heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
7. Incubate the cells on ice for 5 minutes.
8. Add 200 μl of SOC media (make sure that the SOC does not contain antibiotics and is not contaminated) to each transformation, for a total of 250ul.
9. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
10. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
11. For the control, label two petri dishes with LB agar (with the appropriate antibiotic). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
12. Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
13. You can pick single colonies, do a colony PCR, make a glycerol stock, grow up a cell culture and miniprep.
14. Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.

Other Resources

Video

Transforming Your Part from iGEM Videos.

• Please note, this video may be outdated.