Boston UniversityZymo/SOB
From 2007.igem.org
(Difference between revisions)
(→Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB) |
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- | 0.5 mL fresh overnight E. coli culture. The E. coli culture should be grown at 20-32° C. | + | #0.5 mL fresh overnight E. coli culture. The E. coli culture should be grown at 20-32° C. |
- | Inoculate 50 mL Zymo Broth or SOB in 500 mL culture flask | + | #Inoculate 50 mL Zymo Broth or SOB in 500 mL culture flask |
- | Shake 150-250 rpm at 20-33° C until OD is 0.2-0.6, preferably 0.2 | + | #Shake 150-250 rpm at 20-33° C until OD is 0.2-0.6, preferably 0.2 |
- | Prior to harvesting cells, prepare 5 mL of 1x Wash buffer and Competence buffer by adding 2.5 mL of dilution buffer to 2.5 mL of 2x Wash and Competence buffers. Store 1x buffers on ice prior to use. | + | #Prior to harvesting cells, prepare 5 mL of 1x Wash buffer and Competence buffer by adding 2.5 mL of dilution buffer to 2.5 mL of 2x Wash and Competence buffers. Store 1x buffers on ice prior to use. |
- | Transfer the culture from step 2 to ice for 10 minutes. | + | #Transfer the culture from step 2 to ice for 10 minutes. |
- | Cool centrifuge to 0-4° C by using the “Fast Temp” button, preferably at 2° C. | + | #Cool centrifuge to 0-4° C by using the “Fast Temp” button, preferably at 2° C. |
- | Pellet the cells at 3,000-3,700 rpm at 4° for 10 minutes. | + | #Pellet the cells at 3,000-3,700 rpm at 4° for 10 minutes. |
- | Remove supernatant and resuspend the cells in 5 mL of ice-cold, 1x Wash buffer. | + | #Remove supernatant and resuspend the cells in 5 mL of ice-cold, 1x Wash buffer. |
- | Repellet the cells as in step 6. | + | #Repellet the cells as in step 6. |
- | Completely remove the supernatant and gently resuspend the cells in ice-cold 1x Competence buffer. | + | #Completely remove the supernatant and gently resuspend the cells in ice-cold 1x Competence buffer. |
- | Aliquot on ice 0.1-0.2 mL of the cell suspension into epitubes. | + | #Aliquot on ice 0.1-0.2 mL of the cell suspension into epitubes. |
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- | Pre-warm culture plate by placing in 37° incubator for half an hour before preparing cells. | + | #Pre-warm culture plate by placing in 37° incubator for half an hour before preparing cells. |
- | Add 1-5 microliters of plasmid DNA to a tube of thawed, Z-competent cells on ice. Mix gently for a few seconds (keep the DNA volume less than 5% of the total). Let it sit on ice for 5-10 minutes on ice before step 3. | + | #Add 1-5 microliters of plasmid DNA to a tube of thawed, Z-competent cells on ice. Mix gently for a few seconds (keep the DNA volume less than 5% of the total). Let it sit on ice for 5-10 minutes on ice before step 3. |
- | Add 4 volumes of SOC (for example, 400 microliters to 100 microliters) to the transformation mixture and incubate for 1 hour at 37° C, with gentle shaking at 200-300 rpm. | + | #Add 4 volumes of SOC (for example, 400 microliters to 100 microliters) to the transformation mixture and incubate for 1 hour at 37° C, with gentle shaking at 200-300 rpm. |
- | Spread 50-100 microliters of mixture onto a pre-warmed culture plate containing antibiotic. | + | #Spread 50-100 microliters of mixture onto a pre-warmed culture plate containing antibiotic. |
- | Incubate the plate overnight at 37° C. | + | #Incubate the plate overnight at 37° C. |
Latest revision as of 14:43, 2 July 2007
Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
Preparation of Z-competence cells
Procedure can be completed in one day with O/N cells
- 0.5 mL fresh overnight E. coli culture. The E. coli culture should be grown at 20-32° C.
- Inoculate 50 mL Zymo Broth or SOB in 500 mL culture flask
- Shake 150-250 rpm at 20-33° C until OD is 0.2-0.6, preferably 0.2
- Prior to harvesting cells, prepare 5 mL of 1x Wash buffer and Competence buffer by adding 2.5 mL of dilution buffer to 2.5 mL of 2x Wash and Competence buffers. Store 1x buffers on ice prior to use.
- Transfer the culture from step 2 to ice for 10 minutes.
- Cool centrifuge to 0-4° C by using the “Fast Temp” button, preferably at 2° C.
- Pellet the cells at 3,000-3,700 rpm at 4° for 10 minutes.
- Remove supernatant and resuspend the cells in 5 mL of ice-cold, 1x Wash buffer.
- Repellet the cells as in step 6.
- Completely remove the supernatant and gently resuspend the cells in ice-cold 1x Competence buffer.
- Aliquot on ice 0.1-0.2 mL of the cell suspension into epitubes.
- Steps 4-11 must be done at 0-4° C.
Transformation of Z-competent cells (this is for non-ampicillin selection)
Procedure can be completed in one day with O/N cells
- Pre-warm culture plate by placing in 37° incubator for half an hour before preparing cells.
- Add 1-5 microliters of plasmid DNA to a tube of thawed, Z-competent cells on ice. Mix gently for a few seconds (keep the DNA volume less than 5% of the total). Let it sit on ice for 5-10 minutes on ice before step 3.
- Add 4 volumes of SOC (for example, 400 microliters to 100 microliters) to the transformation mixture and incubate for 1 hour at 37° C, with gentle shaking at 200-300 rpm.
- Spread 50-100 microliters of mixture onto a pre-warmed culture plate containing antibiotic.
- Incubate the plate overnight at 37° C.