Difference between revisions of "PhiX:Transformation"
From CCGB
| Line 16: | Line 16: | ||
* discard supernatent | * discard supernatent | ||
* using frosty 1ml tip, resuspend each pellet with 500ul ice-cold 0.1M CaCl<sub>2</sub> (do not vortex) | * using frosty 1ml tip, resuspend each pellet with 500ul ice-cold 0.1M CaCl<sub>2</sub> (do not vortex) | ||
| + | |||
| + | 'At this point may store cells at +4<sup>o</sup>C for 24-48 hours - increases efficiency.' | ||
| + | 'For long-term storage: snap freeze in liquid nitrogen.' | ||
| + | |||
* using frosty 200ul tip, transfer 100ul aliquots to (thin-walled) PCR tubes | * using frosty 200ul tip, transfer 100ul aliquots to (thin-walled) PCR tubes | ||
* add DNA (e.g., 5ul mutagenesis PCR) to each 100ul aliquot | * add DNA (e.g., 5ul mutagenesis PCR) to each 100ul aliquot | ||
Revision as of 16:55, 24 August 2009
With credit to Art Poon for base protocol and Maniartis.
- pre-chill centrifuge to 4oC 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 OD600nm = 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 4oC
- discard supernatent - pellets should be thumb-nail size
- using 10ml pipette, dispense 5ml ice-cold 0.1M CaCl2 to each tube
- resuspend pellet (Art Poon says do not disrupt pellet)
- chill cells on ice for 30 mins
- centrifuge @ 2700g for 10 minutes at 4oC - 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 +4oC for 24-48 hours - increases efficiency.' 'For long-term storage: snap freeze in liquid nitrogen.'
- using frosty 200ul tip, transfer 100ul aliquots to (thin-walled) PCR tubes
- add DNA (e.g., 5ul mutagenesis PCR) to each 100ul aliquot
- chill on ice for 1 hour (meanwhile heat waterbath to 42oC)
- transfer tubes to waterbath, incubate for 1 minute and return tubes to ice
- recovery in SOC is optional
- transfer aliquots to soft agar with 100ul O/N C122 and MgCl/CaCl solution for infection and plate
- incubate plates for four hours at 37oC (Art Poon: 33oC)
