Resuspend and amplify parts from iGEM distribution kits. Adapted from the various distribution handbooks published by iGEM. Note that each part comes as a plasmid, not linear fragments.
A. Locate desired parts on a plate
Use the following platemaps to identify which plate and well contains a desired part.
Make a note of the following properties. Note you should already know what enzymes and fusion sites are used with each part.
Resistance marker
Kit plate (if applicable)
Well
For good practice, also save the full plasmid sequences of each part to be retrieved on SnapGene to simulate assembly.
B. Gather materials
Materials
Competent E. coli cells, kept on ice
Molecular water
Two LB agar plates with appropriate antibiotic from step A2 per part retrieved
Sterile spreader or glass beads
LB or SOC medium
Floating foam tube rack
Microcentrifuge tubes
Desired distribution kit plate
One culture tube or flask per part retrieved
Equipment
Water bath at 42C
Microcentrifuge
Bunsen burner
Timer
Ice bucket
C. Resuspend DNA
If this is the first time a part is being retrieved, follow these steps. Otherwise, simply thaw the distribution kit and take 1 uL of resuspension.
Mark a desired well on the distribution kit with a permanent marker. A plate is oriented like so:
Use a pipette tip to carefully pierce the well and discard the tip. Do not peel off the foil cover.
Pipette 10 uL of molecular-grade water into a well and pipette up and down several times to resuspend, then let it sit for ~5 minutes. The resuspension should look red because it has cresol red dye.
Place the plate(s) in the -20C freezer with the lid closed. There is no need to wrap the plates. The resulting resuspension has a concentration of 100-200 pg/uL DNA.
D. Transform into cells
Label and pre-chill centrifuge tubes on ice. Ensure the water bath is ready at 42C.
Pipette 50 uL of competent cells per tube and keep on ice.
Pipette 1 uL of reuspended DNA into its corresponding centrifuge tube with comp cells. Close tubes and flick gently to mix, but do not vortex.
Incubate tubes on ice for 30 mins.
Using a floating tube rack, heat shock tubes in the 42C water bath for 1 min. Rest tubes on ice for 5 mins.
Pipette 200 uL LB or SOC medium (no AB needed) into each tube.
Incubate tubes in the 37C room for 2 hours, preferably on a shaker or rotor.
Pipette 20 uL onto a corresponding plate and spread immediately with a sterile loop or glass beads. Label the plate with appropriate information, including the inocculation volume.
Repeat step D8 on a second plate, this time with 200 uL.
Incubate plates at 37C overnight and continue the following day with liquid culture, to prep DNA or make glycerol stocks.
