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Team:Newcastle/9 July 2010
From 2010.igem.org
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===Transformation=== | ===Transformation=== | ||
| - | Tubes used: | + | ====Tubes used:==== |
| - | Each of the following tubes contain 200 µl of competent ''E. coli'' DH5alpha. To this the | + | Each of the following five tubes contain 200 µl of competent ''E. coli'' DH5alpha. To this the DNA to be transformed was added. |
# 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3) | # 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3) | ||
# 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5) | # 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5) | ||
| Line 9: | Line 9: | ||
# Control for transformation (with plasmid, pSB1AT3) | # Control for transformation (with plasmid, pSB1AT3) | ||
| - | Protocol: | + | ====Protocol:==== |
# Thaw a 200 µl aliquot of ''E. coli'' DH5alpha. Add the transforming DNA. We added 5 µl of vectors from tubes 1 to 3 and 1 µl 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. | # Thaw a 200 µl aliquot of ''E. coli'' DH5alpha. Add the transforming DNA. We added 5 µl of vectors from tubes 1 to 3 and 1 µl 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. | ||
Revision as of 14:28, 21 July 2010
Transformation
Tubes used:
Each of the following five tubes contain 200 µl of competent E. coli DH5alpha. To this the DNA to be transformed was added.
- 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
- 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
- Negative control for ligation (contains vector with no insert)
- Control for transformation (without plasmid)
- Control for transformation (with plasmid, pSB1AT3)
Protocol:
- Thaw a 200 µl aliquot of E. coli DH5alpha. Add the transforming DNA. We added 5 µl of vectors from tubes 1 to 3 and 1 µl 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 mins on ice.
- Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
- Add 1 ml 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.
