From 2011.igem.org
INP cloning
Project: Surface Display
Date: Wed, 13 Jul 2011 16:20:04 GMT
Author: Viki Ding (dingx055)
Access: Public
Revision History:
- Mon, 26 Sep 2011 16:27:33 GMT (dingx055): entry created in project'Surface Display' by dingx055 (id=9)
Objective: ligate INP into pMCS5BB, perform tranformation.
Ligative 30uL reactions
vector to fragments ratio = 1:4
- vector: 2 uL
- INP (6.09 ug/mL): 8 uL
- ligase: 1 uL
- ligase buffer: 3 uL
- H2O: 16 uL
Negative control
- vector: 6 uL
- H2O: 20 uL
- ligase: 1 uL
- ligase buffer: 3 uL
Incubate at 16 degrees for 1 Hr.
Positive control >> 2 uL of pMCS5BB plasmids uncut
Transformation protocol:
- use competent cells BL21
- add 40 uL BL21 to ligation rxn tubes, incubate on ice for 30 min
- 2 min heat shock at 42 degrees
- cool on ice for 1 min
- immediately add 600 uL of recovery media
- incubate in 37 degrees shaker for 1 hr
- pellet cells, discard excess media
- re-suspend cells in remaining media, plate on gent plates
Results: growth on all plates. Did not purify digestions before ligation.
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