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Revision as of 21:23, 17 October 2007 (view source) Joyxi (Talk | contribs) ← Older edit		Revision as of 17:53, 25 October 2007 (view source) Joyxi (Talk | contribs) Newer edit →
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-	'''Electroporation (Using Bio-Rad Gene Pulser):'''	+	'''Overexpression'''
-	1. Warm up LB + antibiotic plates in 37°C incubator.	+	1. Add 50 ml media (LB or minimal media) and 50 ul antibiotic (Carbenicillin) into flask.
-	2. Thaw bacterial cells on ice. Place sterile cuvettes on ice.	+	2. Innoculate cells and incubate in 37°C shaker overnight.
-	3. Mix 1-2 µl of DNA with the cells.	+	3. Use UV/Vis spectroscopy to measure OD value.
-	4. Set up the Gene Pulser apparatus.	+	4. Add enough culture into new flask to get a final OD of 0.5 and final volume of 50 ml by adding new media.
-	5. Transfer the DNA-cell mixture to the middle of a cold electroporation cuvette. Place in the cuvette chamber slide, and push the slide into the chamber to make contact with the electrodes. Pulse once per sample. Remove the cuvette and immediately add 1 ml LB broth and resuspend the cells.	+	5. Add 1µM Isopropyl β-D-thiogalactopyranoside and incubate flask in 37°C shaker
-	6. Record the time and voltage pulse parameters for each sample. The time constant should be between 3.2-6.0 msec.	+	6. After 24 hours, harvest the cells and store the cell pellet at - 80ْC
-		+
-	7. Transfer the cell suspension to a disposable falcon tube and incubate 30°C for 1 hour while shaking.	+
-		+
-	8. Keep 50 uL of cell and spin down the other 950 uL of cell to obtain a pellet.	+
-		+
-	9. Dissolve the pellet in 50 uL LB.	+
-		+
-	10. Plate both 50 uL of cells on separate, warmed LB plate with antibiotics.	+
-		+
-	11. Incubate overnight in 37°C with plate upside down	+

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Revision as of 17:53, 25 October 2007

Overexpression

1. Add 50 ml media (LB or minimal media) and 50 ul antibiotic (Carbenicillin) into flask.

2. Innoculate cells and incubate in 37°C shaker overnight.

3. Use UV/Vis spectroscopy to measure OD value.

4. Add enough culture into new flask to get a final OD of 0.5 and final volume of 50 ml by adding new media.

5. Add 1µM Isopropyl β-D-thiogalactopyranoside and incubate flask in 37°C shaker

6. After 24 hours, harvest the cells and store the cell pellet at - 80ْC