Team:Osaka/Meeting

From Notebook

=Material and Methods=

Preparing CaCl2competent cell

 * Pick a single colony (strain -> Nova Blue) and inoculate 5 ml LB
 * Incubate overnight at 37°C
 * Inoculate 30 ml LB with 1ml of o/n preculture in sterile 100 ml erlenmeyer
 * Incubate culture at 37°C on a shaker up to OD600=0.3~0.5 (Measure OD value first 1hr and each 30min)
 * When the culture reaches an OD600 between 0.3 and 0.5, transfer the culture into 50 centrifugation tube(sterile)
 * On ice for 10min
 * Centrifuge:8krpm,5min,4°C
 * Discard supernatant
 * Resuspend each pellet in 20 ml chilled 0.1 M MgCl2 and add further 15 ml 0.1 M MgCl (total 45 ml)
 * On ice for 10min
 * Centrifuge:8krpm,5min,4℃
 * Discard supernatant
 * Resuspend each pellet in 20 ml chilled 0.1 M CaCl2 and add further 15 ml 0.1 M MgCl (total 45 ml)
 * On ice 30min
 * Centrifuge:8krpm,5min,4℃
 * Discard supernatant carefuly
 * Resuspend each pellet in 750 µl pre-chilled 0.1 M CaCl2 and 750 µl pre-chilled 50%(v/v) Glycerol
 * Aliquot 500~100 ul of the mix into sterile microfuge tubes that are pre-chiled at -80℃
 * stock at -80℃

Transformation of chemical competent cell

 * Thaw competent cells on ice
 * Mix 2~5 ul pDNA and 100 ul competent cells in 1.5 ml pre-chiled sterile tube
 * On ice for 30min
 * Heat shock 42℃ for 2~1min
 * On ice for 5min
 * Add 900 ul LB
 * Incubate at 37℃ for 1hour (During this, warm plates at 42℃)
 * Harvest cells by centrifuge (14krpm, 1min, r.t.)
 * Discard 900~800 ul of supernatant
 * Resuspend pellet by pipetting
 * Plating all cells on the plate with appropriate antibiotics by glass beads
 * Incubate overnight at 37°C
 * Watch the plate and find colony

Plasmid mini prep (QIAprep Spin Miniprep kit)

 * Pick a single colony from transformed cells and inoculate 5 ml LB with the appropriate selective antibiotics (Amp->5 ul, Kan->25 ul)
 * Incubate overnight at 37°C
 * Harvest the cells by centrifugation 13krpm, 1min, r.t.
 * Resuspend pelleted cells in 250 ul Buffer P1. No cell clumps should be visible after resuspension of the pellet. So, pipetting an vortex well!.P1 buffer must be stocked at 4°C
 * Add 250 ul Buffer P2 and inventing the tubes 4-6times
 * Add 350 ul Buffer N3 and inventing the tubes 4-6times soon after step 5
 * Centrifuge:15krpm, 10min, 4°C
 * Transfer the supernatants to the Ecospin column (economical..) or QlAprep spin column by pipetting
 * Centrifuge: 13krpm, 1min, r.t.
 * Reapply the flow-through to column for improving yield
 * Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
 * Add 500 ul Buffer PB in column
 * Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
 * Add 750 ul Buffer PE in column
 * Centrifuge: 13krpm, 1min, r.t. and discard the flow-through
 * Centrifuge: 14krpm, 3min, r.t. to remove residual wash buffer. And then, discard the flow-through
 * Place the column in a clean 1.5 ml tube. Add 100~50 ul Buffer EB or sterile water to the center of each column
 * Keep at r.t. for 3~5min
 * Centrifuge:15krpm, 1min
 * stock -30°C

Restriction Digestion
For cloning experiments, the final volume should be 20 or 50