Difference between revisions of "PhiX:Transformation"
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* cool for 5 minutes on ice (Maniartis says 10 min) | * cool for 5 minutes on ice (Maniartis says 10 min) | ||
* centrifuge @ 2700g for 10 minutes at 4<sup>o</sup>C | * centrifuge @ 2700g for 10 minutes at 4<sup>o</sup>C | ||
| − | * discard supernatent - pellets should be | + | * discard supernatent - pellets should be pinky-nail size |
* using 10ml pipette, dispense 5ml ice-cold 0.1M CaCl<sub>2</sub> to each tube | * using 10ml pipette, dispense 5ml ice-cold 0.1M CaCl<sub>2</sub> to each tube | ||
| − | * resuspend pellet | + | * resuspend pellet |
* chill cells on ice for 30 mins | * chill cells on ice for 30 mins | ||
* centrifuge @ 2700g for 10 minutes at 4<sup>o</sup>C - pellets should be ring-shaped | * centrifuge @ 2700g for 10 minutes at 4<sup>o</sup>C - pellets should be ring-shaped | ||
| Line 21: | Line 21: | ||
* 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 or 1/3 ligation reaction) to each 100ul aliquot |
* chill on ice for 1 hour (meanwhile heat waterbath to 42<sup>o</sup>C) | * chill on ice for 1 hour (meanwhile heat waterbath to 42<sup>o</sup>C) | ||
| − | * transfer tubes to waterbath, incubate for 1 minute | + | * 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<sup>o</sup>C for 45 minutes or fewer'' | ||
| + | * spread plates with cell/DNA(/SOC) mixture and incubate O/N at 37<sup>o</sup>C | ||
| + | |||
| + | '''Alternatively if plating mutagenic phage:''' | ||
* transfer aliquots to soft agar with 100ul O/N C122 and MgCl<sub>2</sub>/CaCl<sub>2</sub> solution for infection and plate | * transfer aliquots to soft agar with 100ul O/N C122 and MgCl<sub>2</sub>/CaCl<sub>2</sub> solution for infection and plate | ||
| − | * incubate plates for four hours at 37<sup>o</sup>C | + | * incubate plates for four hours at 37<sup>o</sup>C |
Latest revision as of 12:00, 29 June 2013
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 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 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: 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 42oC)
- 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 37oC for 45 minutes or fewer
- spread plates with cell/DNA(/SOC) mixture and incubate O/N at 37oC
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 37oC
