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29/07/13
From 2013.igem.org
(Difference between revisions)
(→Ran the gel from the 26/07/2013 again for 45min) |
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==Ran the gel from the 26/07/2013 again for 45min== | ==Ran the gel from the 26/07/2013 again for 45min== | ||
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==Digestion of plasmid backbone (pSB1C3)== | ==Digestion of plasmid backbone (pSB1C3)== | ||
Revision as of 15:18, 29 July 2013
Contents |
Ran the gel from the 26/07/2013 again for 45min
Digestion of plasmid backbone (pSB1C3)
- Three different reactions
- Master Mix (changes were made from the protocol)
- 5ul NEB Buffer 3.1
- 0.5ul of EcoRI
- 0.5ul of PstI
- 19ul of dH2O
- For each reaction add:
- 4ul of linearized backbone
- 4ul of enzyme master mix
- digest at 37C for 30min
- heat kill at 80C for 20min
Digestion of the limonene biobrick plasmid backbone (BBa_K118025)
- Using clone 1.1 purified plasmid DNA
- For the reaction adding:
- 5ul NEB Buffer 3.1
- 2ul of EcoRI
- 2ul of PstI
- 2.5ul of dH2O
- 38.5ul of DNA
- digest at 37C for 30min
- heat kill at 80C for 20min
Ligation of insert to vector
- Calculate appropriate vector:insert ratio and convert to a 1:1 ratio
- Add the following substances
- 11.1ul of dH2O
- 1ul of QS ligase
- 5ul of 4xQS buffer (vortex before use)
- mix thoroughly by pipetting
- incubate at room temperature for 5 min to create cohesive ends
- run 2.5-5ul of the ligation mixture onto an agarose gel to check ligation efficiency
- transform it into competent cells (E.coli)
