Team:University of Chicago/Notebook/Transformations

General transformation

 * 1) Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
 * 2) * This is at 10 pg/microliter or 10-5 _g/_l
 * 3) * This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
 * 4) Hold on ice 0.5 hours
 * 5) Heat shock 60 sec at 42C
 * 6) Add 250 _l SOC
 * 7) Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
 * 8) * using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
 * 9) * For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
 * 10) * Ampicillin and kanamycin appear to do fine with 1 hour growth
 * 11) Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
 * Good cells should yield around 100 - 400 colonies
 * Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
 * We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

iGEM transformation (using paper spots)
Materials needed Procedure
 * Spots soaked in 5microliters warm TE for 20 minutes
 * 2.0 conical bottom tubes
 * Ice
 * Competent cells
 * 42C water bath/37C incubator
 * SOC--freshly made!
 * Petri plates with appropriate antibiotic
 * Transformaiton control DNA, like pGreen
 * 1) Soak the spots in 5microliters 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 lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation.
 * 3) *Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum
 * 4) Hold the DNA and competent cells on ice for 30 mintues.
 * 5) Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.
 * 6) Incubate cell son ice for 2 minutes
 * 7) Add 200microliters of SOC broth. This should have NO antibiotics.
 * 8) Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.
 * 9) LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.
 * 10) Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, especially ampicillin to break down and untransformed cells will grow.