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29/07/13
From 2013.igem.org
(Difference between revisions)
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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 | ||
#Digestion of plasmid backbone (pSB1C3) | #Digestion of plasmid backbone (pSB1C3) | ||
| + | |||
*Three different reactions | *Three different reactions | ||
*Master Mix (changes were made from the protocol) | *Master Mix (changes were made from the protocol) | ||
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*digest at 37C for 30min | *digest at 37C for 30min | ||
*heat kill at 80C for 20min | *heat kill at 80C for 20min | ||
| - | + | ||
| + | #Digestion of the limonene biobrick plasmid backbone (BBa_K118025) | ||
*Using clone 1.1 purified plasmid DNA | *Using clone 1.1 purified plasmid DNA | ||
*For the reaction adding: | *For the reaction adding: | ||
Revision as of 14:59, 29 July 2013
- 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)
