User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/11: 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"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==12/11/12== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Note: colonies from retransformation of pSB1A3-RFP look red | ||
* | * Assembly: KAH111 + C-His (trouble shooting for Behzad, start from plasmids) | ||
* Minipreps: Rene's pSB1A3-RFP (2); cut with X/S for Brady and Vi's Gibson cloning | |||
---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
# | # KAH111/C-His: 5' part/(E/P)/1123 + C-His/(E/P)/3600 | ||
* Digests (Fermentas FD) | * Digests (Fermentas FD) | ||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest rxn. table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH121112_gel2.png|200px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA (plasmid) || | | DNA (plasmid) || 15.0 μL | ||
|- | |- | ||
| 10x buffer || 3.0 | | 10x buffer || 3.0 | ||
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| enzyme 2 || 1.0 | | enzyme 2 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 10.0 | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~30 min. | | || 30 μL --> 37°C/ ~30 min. | ||
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* Measure conc.'s | * Measure conc.'s of DNA purified from gel (Zymo kit) | ||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 1. KAH11 (E/P) || 0.019 || 1.96 || 18.7 | ||
|- | |- | ||
| 2. | | 2. C-His (E/P) || 0.047 || 1.82 || 46.8 | ||
| | |||
|} | |} | ||
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{| {{table}} cellspacing="3" <!-- Ligations table --> | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> | | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 12/12/12</font> | ||
|- | |- | ||
| 1. | | 1. KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 25ng || <font color="blue">KAH111/C-His 3:1 (Pick 4)</font> (very small colonies after ~18 hours) | ||
|- | |- | ||
| 2. | | 2. C-His(E/P)/ 25 ng || 3 colonies | ||
|} | |} | ||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| || 1 || 2 | | || 1 || 2 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 1.0 || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 0.5 || 0.5 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 2.5 || 3.5 | ||
|- | |- | ||
| || | | || 10 μL || 10 μL | ||
|} | |} | ||
---- | ---- | ||
''' | '''Minipreps'''<br> | ||
* pSB1A3-RFP (retransformation from 12/10/12) | |||
* Note: pellets are red | |||
* Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi) | |||
* Note: combined mini preps afterwards; concentration = 60 ng/μL | |||
{| | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
| | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>3. pSB1A3 = 2155<br>4. pSB1A3 = 2155<br> | |||
| rowspan="7" | [[Image:KAH121112_gel1.png|200px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| | | DNA(plasmid) || 10.0 μL | ||
|- | |- | ||
| | | 10X buffer || 3.0 | ||
|- | |- | ||
| || | | XbaI || 1.0 | ||
|- | |||
| SpeI || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 15.0 | |||
|- | |||
| || 15 μL --> 37°C/ ~30 min. | |||
|} | |} | ||
* Gel purification: combined the two slices | |||
** Concentration: 260 = 0.015, 260/280 = 2.2, '''ng/μL = 15.1''' | |||
Latest revision as of 22:19, 26 September 2017
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12/11/12
Assemblies
Minipreps
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