User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22: 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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== | ==05/22/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Ryan - Bra minipreps & digests | ||
* | * Cas-tone - minipreps & digests | ||
---- | ---- | ||
''' | '''Ryan - Bra minipreps & digests''' | ||
* Check with | |||
* Minipreps<br> | |||
** Sigma GenElute kit, elute w/ 75 μL elution sln. | |||
* Digests | |||
** Check with EcoRI digests - promoter insertion generates a new EcoRI site | |||
** Note: BraR = 776, pBra = 214 | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <u>Expected:</u><br>1. | | rowspan="7" | <u>Expected:</u><br>1-8. pBra-BraR_MRV = 2989, 1202<br>empty vector = 3977<br> | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH052215_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA(plasmid) || 2.0 μL | | DNA(plasmid) || 2.0 μL | ||
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| EcoRI || 1.0 | | EcoRI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 10.5 | |||
| dH<sub>2</sub>O || | |||
|- | |- | ||
| || 15 μL | | || 15 μL | ||
|} | |} | ||
--> 37°C/ ~15 min. | |||
---- | ---- | ||
''' | '''Cas-tone - H3_MV11 minipreps & digests''' | ||
* Cultures | |||
** Pick 2 colonies from each ligation plate | |||
** Make streak library & 5 mL cultures | |||
* Digests | * Digests | ||
** | ** Check with S/HindIII digests - H3 insertion destroys SpeI site | ||
** Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band | |||
** H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433 | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | < | | rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV11 = 2345, 7291<br>3,4. H3-1-60_MV11 = 2345, 7351<br>5,6. H3-1-80_MV11 = 2345, 7412<br>7,8. H3-1-116_MV11 = 2345, 7535<br>9,10. H3-1-136_MV11 = 2345, 7595<br> | ||
| rowspan="7" | [[Image:KAH052215_gel2.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| DNA (plasmid) || | | DNA(plasmid) || 2.0 μL | ||
|- | |- | ||
| | | 10X buffer || 1.5 | ||
|- | |- | ||
| | | SpeI || 1.0 | ||
|- | |- | ||
| | | HindIII || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 9.5 | ||
|- | |- | ||
| || 15 μL | |||
| | |||
|} | |} | ||
--> 37°C/ ~15 min. | |||
Latest revision as of 00:59, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||
05/22/15
Ryan - Bra minipreps & digests
--> 37°C/ ~15 min.
Cas-tone - H3_MV11 minipreps & digests
--> 37°C/ ~15 min.
|