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===Heat-shock transfection=== *Equilibrate water bath to 42C. *Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min. *Thaw the TOP10 E.coli competent cell vial on ice. *Transfer '''1uL''' ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. ('''Mixing by repeat pipetting is prohibited in the protocol'''). *Set up competent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins. *Adjust the timer: set up 30sec and 2min. *Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min. *Add 250ul of SOC medium into each vial (drop the SOC solution slowly throw the tube wall). *Shake the vials horizontally at 37C for 1 hour using vortex on 3rd floor at the lowest speed. *During the 1hr incubation. *After 1hr shaking, plate '''50uL''' from each transformation vial on agar plates (w/ Kanamycine). **There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design. *Incubate plates at 37C for about 20hr. <6pm 11/18/10 -> 4pm 11/19/10> '''Pick up 10 colonies per sample''' *Prepare LB medium with 10% glycerol and 35ug/ml Kanamycin. *200ul per well [[File:Bis-PCR Progeria - plate layout.png]]
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