Objective
- Try electroporation
- Re-ligate with different I:V
Bench work
- Electrocompetent DH10B
- t=0 OD600=0.08
- t~1.5h OD600~0.2
- t~2h OD600~0.6 → stop growth
- incubate culture ~10' on ice w/ swirling
- spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C Sorvall RC6+ and SH-3000 rotor
- supernatent
- pellet
- resuspend in 100 mL cold sterile H2O
- spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C
- supernatent
- pellet
- resuspend in 45 mL cold sterile H2O (should be 50 mL, but not enough sterile water)
- spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C
- supernatent
- pellet
- resuspend in 10 mL cold sterile 10% glycerol
- spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C
- supernatent
- pellet
- resuspend in 200 μL cold sterile 10% glycerol
- aliquot into 5 x 50μL (*Kathryn Muratore 15:34, 8 July 2011 (EDT): I meant to freeze the 5th unused aliquot, but did not)
- Transformation by electroporation
- Used David Carlini's Bio-Rad electroporator in Biology
- 0.1 cm cuvettes
- 50μL DH10B + 20μL V(His)+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
- 50μL DH10B + 20μL V(His) neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
- 50μL DH10B + 20μL V+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
- 50μL DH10B + 20μL V neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
- 1.5 kV, t = 3.6-4.0 ms
- add 450μL SOC
- → 1.5h @ 37°C w/ shaking (should be 45')
- plate 100μL of 1:1 for all samples on LBAmp100
- plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp100
- → 37°C O/N
- Re-plate transformants
- spread remaining 450μL of NovaBlue transformations from yesterday
- Ligation
- 8.5μL ddV(His) from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
- 8.5μL ddV from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
Results
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