User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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| colspan="2"| | | colspan="2"| | ||
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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 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 | ||
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| || 10.0 || 10.0 || || 10.0 || 10.0 || | | || 10.0 || 10.0 || || 10.0 || 10.0 || | ||
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* | * 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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Latest revision as of 22:29, 26 September 2017
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02/23/13
Type IIS Assembly, PCR: LOV-H2B+his
PCR
Golden Gate Reactions
Thermal cycling
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