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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==02/23/13== | ==02/23/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Type IIS Assembly, PCR: LOV | * Type IIS Assembly, PCR: H2B-LOV+his | ||
---- | ---- | ||
'''Type IIS Assembly, PCR: LOV-H2B+his''' | '''Type IIS Assembly, PCR: LOV-H2B+his''' | ||
# H2B = 378 bp, | # H2B = 378 bp, 1A3/H2B fwd + H2B/LOV rev (Template: H2B-GFP from Addgene) | ||
# LOV = 429 bp, | # LOV = 429 bp, LOV fwd + LOV+his/1A3 rev (Template: LOV from Addgene); note: reverse primer adds a 6-his tag and TAA stop (21 bp) | ||
# pSB1A3 = ~2000, gg0001/ | # pSB1A3 = ~2000, gg0001 v2 + gg0002 v2 | ||
* Be sure to use the H2B and LOV primers ordered on 2/4/2003. The names are redundant with a previous version. | |||
{| {{table}} border="1" cellspacing="3" <!-- | PCR | ||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
{| {{table}} border="1" cellspacing="3" <!-- PCR rxn. table --> | |||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. H2B = | | rowspan="7" | <u>Expected:</u><br>1. H2B = 378<br>2. LOV = 450<br>3, 4. pSB1A3 = ~2000 | ||
| rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | | rowspan="7" | [[Image:KAH022313_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| 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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| || 50.0 μL | | || 50.0 μL | ||
|} | |} | ||
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| bgcolor=#cfcfcf | ng/μL | | bgcolor=#cfcfcf | ng/μL | ||
|- | |- | ||
| 1. | | 1. H2B || 0.04 || 1.667 || 40.4 | ||
|- | |||
| 2. LOV+his || 0.019 || 1.562 || 18.6 | |||
|- | |- | ||
| | | 3. pSB1A3 || 0.069 || 1.754 || 69.4 | ||
|- | |- | ||
| | | 4. pSB1A3 || 0.085 || 1.817 || 85.5 | ||
|} | |} | ||
Dilutions: to final concentration of 20 fmol/μL | Dilutions: to final concentration of 20 fmol/μL (40 μL) | ||
* x = length in bp ÷ measured ng/μL * 0.013 * 40 | |||
{| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | {| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | ||
|- valign="top" | |- valign="top" | ||
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| bgcolor=#cfcfcf | length (bp) | | bgcolor=#cfcfcf | length (bp) | ||
| bgcolor=#cfcfcf | ng/μL | | bgcolor=#cfcfcf | ng/μL | ||
| bgcolor=#cfcfcf | Vol. | | bgcolor=#cfcfcf | Vol. (x) | ||
| bgcolor=#cfcfcf | + dH<sub>2</sub>O | | bgcolor=#cfcfcf | + dH<sub>2</sub>O | ||
|- | |- | ||
| pSB1A3 || 2000 || | | pSB1A3 || 2000 || 85.5 || 12.2 || 27.8 | ||
|- | |- | ||
| | | H2B || 378 || 40.4 || 4.9 || 35.1 | ||
|- | |- | ||
| | | LOV+his || 450 || 18.6 || 12.6 || 27.4 | ||
|} | |} | ||
(02/25/13)<br> | |||
Golden Gate Reactions | Golden Gate Reactions | ||
# | # H2B + LOV+his + pSB1A3 | ||
# pSB1A3 | # pSB1A3 | ||
# H2B + LOV+his + pSB1A3, 2xDNA | |||
# pSB1A3, 2xDNA | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| Reagent || 1 || 2 | | 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 ligase buffer || 1.0 || 1.0 | | 10x PRO ligase buffer || 1.0 || 1.0 || 3.0 || 1.0 || 1.0 || 3.0 | ||
|- | |- | ||
| NEB T4 lgase || 0.25 || 0.25 | | NEB T4 lgase || 0.25 || 0.25 || 0.75 || 0.25 || 0.25 || 0.75 | ||
|- | |- | ||
| BsmBI || 0.5 || 0.5 | | BsmBI || 0.5 || 0.5 || 1.5 || 0.5 || 0.5 || 1.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || 5.25 || | | dH<sub>2</sub>O || 5.25 || 5.25 || 15.75 || 2.25 || 2.25 || 6.75 | ||
|- | |- | ||
| || 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 | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
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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