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Team:Cambridge/Notebook/Week4
From 2010.igem.org
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===Theo's plan=== | ===Theo's plan=== | ||
# Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol | # Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol | ||
| - | # Digest luciferase Biobrick with: XbaI, PstI | + | # Digest luciferase Biobrick with: XbaI, PstI to produce two fragments: |
| - | # Digest backbone Biobrick with SpeI, PstI | + | ## P end---old backbone---E--Xend |
| + | ## Xend--luciferase---S--Pend <strong>(desired)</strong> | ||
| + | # Digest backbone Biobrick with SpeI, PstI to produce: | ||
| + | ## Send--short fragment--Pend | ||
| + | ## Pend---backbone--E--X--promoter--RBS--Send <strong>(desired)</strong> | ||
# Run both digest products on a gel | # Run both digest products on a gel | ||
# Extract suitable lengthed fragments from gel: | # Extract suitable lengthed fragments from gel: | ||
| Line 32: | Line 36: | ||
## Luciferase should be 1653 bp (its 3426 bp backbone should be discarded) | ## Luciferase should be 1653 bp (its 3426 bp backbone should be discarded) | ||
# Perform ligation | # Perform ligation | ||
| + | ##P and X are compatible so: we get Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend | ||
| + | ##The two P ends will also ligate, producing a circular desired plasmid | ||
# Transform competent TOP10 cells with ligation product | # Transform competent TOP10 cells with ligation product | ||
Revision as of 18:18, 4 August 2010
Notebook: Week 4
Monday
- Wet work
- Transformed competent cells using plasmid phK724 from Jim Slock (thanks!)
- Grew up E. coli containing phK555 in 5ml LB broth in rotating 37°C incubator overnight
- Dry work
- Finalising sequencing requirements
- Dismissed PPK
- Decided to include Luciola cruciata
- Further Wiki work
- Finalising sequencing requirements
Tuesday
- Wet Work
- Made competent cells of 4 different hns mutants: W3110 hns93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10, GM230 hns-205::Tn10
- Now stored in the -80°C freezer for later use
- Dry Work
- Organising sequences for synthesis
Wednesdy
Thursday
Theo's plan
- Miniprep plasmid DNA from luciferase and backbone transformed TOP10 following Qiagen protocol
- Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
- P end---old backbone---E--Xend
- Xend--luciferase---S--Pend (desired)
- Digest backbone Biobrick with SpeI, PstI to produce:
- Send--short fragment--Pend
- Pend---backbone--E--X--promoter--RBS--Send (desired)
- Run both digest products on a gel
- Extract suitable lengthed fragments from gel:
- Backbone should be 2153 bp
- Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)
- Perform ligation
- P and X are compatible so: we get Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend
- The two P ends will also ligate, producing a circular desired plasmid
- Transform competent TOP10 cells with ligation product
Friday
Saturday
Sunday
