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 | ||
Line 55: | Line 55: | ||
| bgcolor=#cfcfcf | ng/μL | | bgcolor=#cfcfcf | ng/μL | ||
|- | |- | ||
| 1. H2B | | 1. H2B || 0.04 || 1.667 || 40.4 | ||
|- | |- | ||
| 2. LOV || 0.019 || 1.562 || 18.6 | | 2. LOV+his || 0.019 || 1.562 || 18.6 | ||
|- | |- | ||
| 3. pSB1A3 || 0.069 || 1.754 || 69.4 | | 3. pSB1A3 || 0.069 || 1.754 || 69.4 | ||
Line 83: | Line 83: | ||
(02/ | (02/25/13)<br> | ||
Golden Gate Reactions | Golden Gate Reactions | ||
Line 95: | Line 95: | ||
| Reagent || 1 || 2 || Master mix (x3) || 3 || 4 || Master mix (x3) | | Reagent || 1 || 2 || Master mix (x3) || 3 || 4 || Master mix (x3) | ||
|- | |- | ||
| | | pSB1A3 gg || 1.0 || 1.0 || --- || 2.0 || 2.0 || --- | ||
|- | |- | ||
| | | H2B gg || 1.0 || H2O || --- || 2.0 || H2O || --- | ||
|- | |- | ||
| | | LOV+his gg || 1.0 || H2O || --- || 2.0 || H2O || --- | ||
|- | |- | ||
| 10x PRO ligase buffer || 1.0 || 1.0 || 3.0 || 1.0 || 1.0 || 3.0 | | 10x PRO ligase buffer || 1.0 || 1.0 || 3.0 || 1.0 || 1.0 || 3.0 | ||
Line 111: | Line 111: | ||
| || 10.0 || 10.0 || || 10.0 || 10.0 || | | || 10.0 || 10.0 || || 10.0 || 10.0 || | ||
|} | |} | ||
* | * For 1 and 2, aliquot 7.0 mix to each tube; add DNA/ H2O | ||
* For 3 and 4, aliquot 4.0 mix to each tube; add DNA/ H2O | |||
Thermal cycling | Thermal cycling | ||
* [45°C, 2 min.; 16°C, 5 min.] x25 | * [45°C, 2 min.; 16°C, 5 min.] x25 | ||
* 60°C, | * 60°C, 10 min. | ||
* 80°C, 20 min. | * 80°C, 20 min. | ||
* 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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