Difference between revisions of "PhiX:Transformation"

Revision as of 16:04, 18 June 2013

With credit to Art Poon for base protocol and Maniartis.

• pre-chill centrifuge to 4^oC and get ice
• freeze two 20ml, two 10ml serological pipettes and 1ml, 200ul tip boxes - use these for all transfers
• chill two 50ml centrifuge tubes
• prepare 50ml LB + 1ml O/N C122 (or chosen strain; Art Poon says inoculate with colony)
• follow until OD_600nm = 0.250 (must catch in exponential phase)
• using 20ml pipette, divide culture into two ~25ml aliquots into chilled centrifuge tubes
• cool for 5 minutes on ice (Maniartis says 10 min)
• centrifuge @ 2700g for 10 minutes at 4^oC
• discard supernatent - pellets should be pinky-nail size
• using 10ml pipette, dispense 5ml ice-cold 0.1M CaCl₂ to each tube
• resuspend pellet
• chill cells on ice for 30 mins
• centrifuge @ 2700g for 10 minutes at 4^oC - pellets should be ring-shaped
• discard supernatent
• using frosty 1ml tip, resuspend each pellet with 500ul ice-cold 0.1M CaCl₂ (do not vortex)

At this point may store cells at +4^oC for 24-48 hours: this increases efficiency.
For long-term storage: snap freeze in liquid nitrogen. Thaw for ten minutes on ice.

• using frosty 200ul tip, transfer 100ul aliquots to (thin-walled) PCR tubes
• add DNA (e.g., 5ul mutagenesis PCR or 1/3 ligation reaction) to each 100ul aliquot
• chill on ice for 1 hour (meanwhile heat waterbath to 42^oC)
• heat shock: transfer tubes to waterbath, incubate for 1 minute exactly
• return tubes to ice for a minute or two
• optional: add cell/DNA mix to 500ul SOC in larger tube and incubate at 37^oC for 45 minutes or fewer
• spread plates with cell/DNA(/SOC) mixture and incubate O/N at 37^oC

Alternatively if plating mutagenic phage:

• transfer aliquots to soft agar with 100ul O/N C122 and MgCl₂/CaCl₂ solution for infection and plate
• incubate plates for four hours at 37^oC (Art Poon: 33^oC)