Name:
Date:
Project:
Heatshock Method!
- 1x 50µL Vial of chemical competent E.coli strain of interest per transformation
- One Shot™ ccdB Survival™ 2 T1R
- Top10
- DB3.1
- XL10-gold
- SHuffle T7
- SHuffle T7 express LysY
- SHuffle express
- SHuffle T7 express
- BL21
- BL21(DE3)
- BL21(DE3) pLysS
- Rosetta2(DE3)
- ArcticExpress(DE3)
- C43(DE3)
- 100ng of plasmid of interest per transformation
- 1x LB-Agar plate with desired selection marker per transformation
- S1 Benchtop Eppi Shaker
- incubator closet
- LB-Medium or SOC Medium
Day 1, date:
- select program 6 on S1 Benchtop Eppi Shaker
- get LB-Agar plate from cold room and leave on bench to get to roomtemp.
- get desired Plasmid from -20°C and thaw on ice
- get E.coli vial from -80°C and thaw on ice for 10min
- add 100ng of plasmid to 1x 50µL vial of E.coli competent cells
- mix by stirring gently with the pipette tip, do not vortex or mix by pipetting up and down
- chill on ice for 10min
- put vial into tabletop shaker and start program 6 (42°C, 45sec, no shaking)
- select program 7 on S1 Benchtop Eppi Shaker
- put vial back on ice until shaker has cooled to 37°C
- add ___________µL LB or SOC Medium to vial (200 – 500µL)
- mix by pipetting
- put into shaker and start program 7 (37°C, 30min, 500 RPM)
- spin down cells in vial
- discard added LB Medium
- resuspend cells in remaining 50µL
- spread on prepared LB-Agar plate with desired selection marker with the help of a drigalski spatula or inoculating loop
- turn upside down and incubate at ______°C for ______h (e.g. 37°C overnight, or 22°C over the weekend)
Day 2, date:
- check LB-Agar plates for colonies
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