From 2007.igem.org

Revision as of 17:53, 25 October 2007 (view source) Joyxi (Talk | contribs) ← Older edit		Latest revision as of 18:04, 25 October 2007 (view source) Joyxi (Talk | contribs)
Line 1:		Line 1:
-	'''Overexpression'''	+	'''Electroporation (Using Bio-Rad Gene Pulser): '''
-	1. Add 50 ml media (LB or minimal media) and 50 ul antibiotic (Carbenicillin) into flask.	+	1. Warm up LB + antibiotic plates in 37°C incubator.
-	2. Innoculate cells and incubate in 37°C shaker overnight.	+	2. Thaw bacterial cells on ice. Place sterile cuvettes on ice.
-	3. Use UV/Vis spectroscopy to measure OD value.	+	3. Mix 1-2 µl of DNA with the cells.
-	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.	+	4. Set up the Gene Pulser apparatus.
-	5. Add 1µM Isopropyl β-D-thiogalactopyranoside and incubate flask in 37°C shaker	+	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.
-	6. After 24 hours, harvest the cells and store the cell pellet at - 80ْC	+	6. Record the time and voltage pulse parameters for each sample. The time constant should be between 3.2-6.0 msec.
		+
		+	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
		+
		+	Retrieved from "https://2007.igem.org/Berkeley_LBL/Electroporation"

---

Latest revision as of 18:04, 25 October 2007

Electroporation (Using Bio-Rad Gene Pulser):

1. Warm up LB + antibiotic plates in 37°C incubator.

2. Thaw bacterial cells on ice. Place sterile cuvettes on ice.

3. Mix 1-2 µl of DNA with the cells.

4. Set up the Gene Pulser apparatus.

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.

6. Record the time and voltage pulse parameters for each sample. The time constant should be between 3.2-6.0 msec.

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

Retrieved from "https://2007.igem.org/Berkeley_LBL/Electroporation"