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From 2012.igem.org

Contents 1 I. Began growing stock RiAFP+GFP DH5-α cells (dissolve some scraped cells in 10 mL LB medium with 10 microliters of both ampicillin and kanamycin) 2 II. Transformed DH5-α cells 3 III. Attended a meeting about primer design with Jimin Wang and Wuyui 4 IV. Began designing primers 5 V. Miniprepped RiAFP+GFP cells grown from morning: (follow all aseptic guidelines like keeping a burning flame near work station)

I. Began growing stock RiAFP+GFP DH5-α cells (dissolve some scraped cells in 10 mL LB medium with 10 microliters of both ampicillin and kanamycin)

II. Transformed DH5-α cells

1. Thaw competent cells on ice
2. Aliquot 100 microliters of cells into 2 pre-chilled 15 mL culture tubes
3. Add DNA (1 to 5 µL), swirl, and incubate on ice for 20 minutes
4. Heat shock for 40 sec at 42°C
5. Add 1 mL SOC
6. Incubate at 37°C shaker for 1 hour
7. Using beads, spread 100microliters on antibiotic plates
8. Grow at 37C overnight to test for competency (about 16 hours to be ready for maniple).

III. Attended a meeting about primer design with Jimin Wang and Wuyui

IV. Began designing primers

V. Miniprepped RiAFP+GFP cells grown from morning: (follow all aseptic guidelines like keeping a burning flame near work station)

1. Spin down/centrifuge about 2 eppendorf tubes 5 times with increments of 1 mL of solution with cells each, draining off the supernatant after each spin and adding more cell solution
2. Resuspend pelleted bacteria in 250 microliters of buffer P1. (the pipet tip may be used to break up the pellets after pipetting the buffer)
3. Add 250 microliters of buffer P2 and mix thoroughly by inverting the tube a few times.
4. Add 350 microliters of buffer N3 and mix immediately and thoroughly by inverting.
5. Centrifuge the eppendorf tubes for at least 10 minutes at 13,000 rpm.
6. Apply as much as possible of the supernatant from step 5 to a spin column without disrupting the pellet. (Pipetting is an option. Also, make the hinges of tubes face up/to the outer radius of centrifuge for easier decanting.)
7. Centrifuge for about a minute and discard the flow through.
8. Wash the spin column by adding .5 mL buffer PB and centrifuge for about a minute + discard flow-through again.
9. Wash the spin column with .75 mL buffer PE and centrifuge for about a minute + discard flow-through again.
10. Centrifuge again for at least a minute more
11. (Eluting DNA) Place the spin column in a sterile 1.5 mL eppendorf tube. Add 30 microliters of water or buffer EB to the spin column. Let stand for at least one minute before centrifuging for about a minute.
12. Add 10 more microliters of water or EB buffer to the spin column. Again, let stand for at least 1 min and centrifuge for at about a min at high speed. (If visible drops still remain in the spin column, try rotating the spin column and centrifuging. Alternatively, slightly lift the column up from the tube, carefully flick it aseptically and so that the rest of DNA solution does not splatter, and centrifuge again.)