"

From 2010.igem.org

Revision as of 14:38, 12 October 2010 (view source) Rchamberlin (Talk | contribs) (New page: == Richard Lab:Electroporation of E. coli == This protocol is for the typical electro-transformation of E. coli done in the Richard Lab. The consensus electroporation protocol should b...) ← Older edit		Latest revision as of 15:11, 12 October 2010 (view source) Rchamberlin (Talk | contribs)
Line 1:		Line 1:
-	== Richard Lab:Electroporation of E. coli ==	+	== '''Preparing SOB''' ==
		+	- SOB BROTH(DRY MEDIA) CatNo. S0221 Lot: S022130D0901
		+	Scale: Mad by Adventurer Pro AV64 s/n: 8030391129 Error+ .67% Calibrated by Cassidy
-	This protocol is for the typical electro-transformation of E. coli done in the Richard Lab.
-	The consensus electroporation protocol should be consulted if deviating from the procedure outlined here. Procedure	+	1) I used 4 - 250 mL capped elenmyer flasks
-	1. Chill the # electroporation cuvettes by floating them in an ice bath.	+	2) in two of them I added 6.97 grams of SOB media powder to 250 mL DI water
-	2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.	+	3)the mixed and dissolved on the hot plate
-	3. Prepare # micro-centrifuge tubes containing 900μl SOB media.	+	4) once dissolved, I separated the 2 - 250 mL SOB media into 4 - 250 mL elenmyer flasks and autocalved for one hour
-		+
-	4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes).	+
-		+
-	5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.	+
-		+
-	6. Place cells back on ice to ensure they remain cold.	+
-		+
-	7. Pippette 100μL of cell-DNA mixture to cuvette.	+
-		+
-	8. Wipe off excess moisture from outside of cuvette.	+
-		+
-	9. Place cuvette in chamber of electroporator.	+
-		+
-	10. Pulse the cells by pressing button on electroporator twice.	+
-		+
-	11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.	+
-		+
-	12. Let cells recover at room temperature for 30-60 minutes.	+
-		+
-	13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.	+
-		+
-	--Rchamberlin 19:59, 30 June 2010 (UTC)	+

---

Latest revision as of 15:11, 12 October 2010

Preparing SOB

- SOB BROTH(DRY MEDIA) CatNo. S0221 Lot: S022130D0901

Scale: Mad by Adventurer Pro AV64 s/n: 8030391129 Error+ .67% Calibrated by Cassidy

1) I used 4 - 250 mL capped elenmyer flasks

2) in two of them I added 6.97 grams of SOB media powder to 250 mL DI water

3)the mixed and dissolved on the hot plate

4) once dissolved, I separated the 2 - 250 mL SOB media into 4 - 250 mL elenmyer flasks and autocalved for one hour