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	==Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB ==		==Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB ==
		+	[http://www.zymoresearch.com/site_search.asp?mode=allwords&search=z-competent&submit.x=0&submit.y=0 Full PDF version]
	Preparation of Z-competence cells		Preparation of Z-competence cells

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Latest revision as of 16:46, 16 July 2007

Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB

[http://www.zymoresearch.com/site_search.asp?mode=allwords&search=z-competent&submit.x=0&submit.y=0 Full PDF version]

Preparation of Z-competence cells

Procedure can be completed in one day with O/N cells

1. 0.5 mL fresh overnight E. coli culture. The E. coli culture should be grown at 20-32° C.
2. Inoculate 50 mL Zymo Broth or SOB in 500 mL culture flask
3. Shake 150-250 rpm at 20-33° C until OD is 0.2-0.6, preferably 0.2
4. 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.
5. Transfer the culture from step 2 to ice for 10 minutes.
6. Cool centrifuge to 0-4° C by using the “Fast Temp” button, preferably at 2° C.
7. Pellet the cells at 3,000-3,700 rpm at 4° for 10 minutes.
8. Remove supernatant and resuspend the cells in 5 mL of ice-cold, 1x Wash buffer.
9. Repellet the cells as in step 6.
10. Completely remove the supernatant and gently resuspend the cells in ice-cold 1x Competence buffer.
11. 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

1. Pre-warm culture plate by placing in 37° incubator for half an hour before preparing cells.
2. 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.
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.
4. Spread 50-100 microliters of mixture onto a pre-warmed culture plate containing antibiotic.
5. Incubate the plate overnight at 37° C.

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