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Duke University/24 June 2009
From 2009.igem.org
(Difference between revisions)
| Line 5: | Line 5: | ||
*R: Correct band (faint) | *R: Correct band (faint) | ||
*C: Do transformation with competent cells | *C: Do transformation with competent cells | ||
| + | |||
| + | Transformation with High Efficiency GC5 Competent Cells | ||
| + | # Remove competent cells from -70°C and place on ice. Thaw for 5-10 min. | ||
| + | # Gently mix cells by tapping tube. | ||
| + | # Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix. | ||
| + | # Place tubes on ice for 30 min. | ||
| + | # Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake! | ||
| + | # Add 450 ul RT SOC Medium to each transformation reaction. | ||
| + | # Incubate at 37°C for 1 hr with shaking at 225-250 rpm. | ||
| + | # Spread on LB agar plates containing appropriate antibiotic. | ||
| + | # Incubate plates at 37°C overnight (12-16 hrs). | ||
Latest revision as of 17:27, 21 October 2009
June 24, 2009
- E: Gel to check PCR
- R: Correct band (faint)
- C: Do transformation with competent cells
Transformation with High Efficiency GC5 Competent Cells
- Remove competent cells from -70°C and place on ice. Thaw for 5-10 min.
- Gently mix cells by tapping tube.
- Add 1-50 ng DNA (1 ul control) into 50 ul cells. Swirl pipette tip while dispensing DNA. Gently tap tube to mix.
- Place tubes on ice for 30 min.
- Heat-shock cells for 45 sec in 42°C (water) bath. Do not shake!
- Add 450 ul RT SOC Medium to each transformation reaction.
- Incubate at 37°C for 1 hr with shaking at 225-250 rpm.
- Spread on LB agar plates containing appropriate antibiotic.
- Incubate plates at 37°C overnight (12-16 hrs).
