User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23: Difference between revisions
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| DNA(plasmid) || 0.2 μL | | DNA(plasmid) || 0.2 μL | ||
|- | |- | ||
| primer 1 || 1.0 | | 10 μM primer 1 || 1.0 | ||
|- | |- | ||
| primer 2 || 1.0 | | 10 μM primer 2 || 1.0 | ||
|- | |- | ||
| 2x GoTaq || 25.0 | | 2x GoTaq || 25.0 | ||
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* 4°C, ∞ | * 4°C, ∞ | ||
Transformation | |||
* Transfer 10 μL reactions into 2.0 mL round bottom tubes; add 50 μL chemically competent BL21 cells (thawed on ice); pipette up and down 3x to mix | |||
* Incubate on ice for 5 min. | |||
* Heat shock at 42°C (heat block) for exactly 45 sec.; place on ice immediately | |||
* Add 750 μL plain LB broth; 37°C incubator: lay tubes flat and tape to the shaking rack; incubate with shaking for 45 min. | |||
* Pellet the cells at top speed for 3 min. at room temp. | |||
* Discard the supernatant; resuspend the pellet in 100 μL LB Amp (100μg/mL); plate on LB Amp agar (100 μg/mL) | |||
* Incubate overnight | |||
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Revision as of 14:29, 26 February 2013
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02/23/13
Type IIS Assembly, PCR: LOV-H2B+his
PCR
Golden Gate Reactions
Thermal cycling
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