Team:Cambridge/Notebook/Week4

From 2010.igem.org

(Difference between revisions)
(Theo's plan)
(Theo's plan)
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# Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
# Digest luciferase Biobrick with: XbaI, PstI to produce two fragments:
## P end---old backbone---E--Xend
## P end---old backbone---E--Xend
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## Xend--luciferase---S--Pend <strong>(desired)</strong>
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## X<sub>end</sub>--luciferase---S--P<sub>end</sub> <strong>(desired)</strong>
# Digest backbone Biobrick with SpeI, PstI to produce:
# Digest backbone Biobrick with SpeI, PstI to produce:
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## Send--short fragment--Pend
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## S<sub>end</sub>--short fragment--P<sub>end</sub>
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## Pend---backbone--E--X--promoter--RBS--Send <strong>(desired)</strong>
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## P<sub>end</sub>---backbone--E--X--promoter--RBS--S<sub>end</sub> <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:
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## 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
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##P and X are compatible so: we get Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend
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##P and X are compatible so: we get P<sub>end</sub>---backbone--E--X--promoter--RBS--SX<sub>scar</sub>--luciferase---S--P<sub>end</sub>
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##The two P ends will also ligate, producing a circular desired plasmid
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##The two P<sub>end</sub>s 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 18:19, 4 August 2010