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Revision as of 11:11, 9 July 2010 (view source) Yessa (Talk | contribs) (New page: ===Transformation=== # 1:3 # 1:5 # Vector (no insert), negative control for ligation # DH5Alpha (without plasmid) # DH5Alpha (with plasmid, pSB1AT3) Protocol: #) ← Older edit		Revision as of 11:21, 9 July 2010 (view source) Yessa (Talk | contribs) Newer edit →
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	Protocol:		Protocol:
-	#	+	# Thaw a 200µl aliquot of E. coli DH5Alpha. Add the transforming DNA. We added 5µl of vectors from tubes 1 to 3 and 1µl of vector from tube 5 because vector  from tube 5 is has not been ligated whereas tubes 1 to 3 have been ligated and will contain cells in different forms.
		+	# Incubate for 45 min on ice.
		+	# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
		+	# Add 1ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
		+	# Plate out 200 µl/plate on LB (agar at 1.5%), with ampicilin.
		+	# Incubate plates overnight at 37°C.

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Revision as of 11:21, 9 July 2010

Transformation

1. 1:3
2. 1:5
3. Vector (no insert), negative control for ligation
4. DH5Alpha (without plasmid)
5. DH5Alpha (with plasmid, pSB1AT3)

Protocol:

1. Thaw a 200µl aliquot of E. coli DH5Alpha. Add the transforming DNA. We added 5µl of vectors from tubes 1 to 3 and 1µl of vector from tube 5 because vector from tube 5 is has not been ligated whereas tubes 1 to 3 have been ligated and will contain cells in different forms.
2. Incubate for 45 min on ice.
3. Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
4. Add 1ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
5. Plate out 200 µl/plate on LB (agar at 1.5%), with ampicilin.
6. Incubate plates overnight at 37°C.