User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/11: Difference between revisions
From OpenWetWare
Line 12: | Line 12: | ||
---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
# | # KAH111/C-His: 5' part/(E/P)/size + C-His/(E/P)/3600 | ||
Line 51: | Line 25: | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | <!-- [[Image: | | rowspan="7" | <!-- [[Image:KAH121112_gel1.png|250px|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 | ||
Line 61: | Line 35: | ||
| 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. | ||
Line 72: | Line 46: | ||
| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 1. KAH11 (E/P) || --- || --- || --- | ||
|- | |- | ||
| 2. | | 2. C-His (E/P) || --- || --- || --- | ||
|} | |} | ||
Line 99: | Line 55: | ||
{| {{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">new BioBrick #:1 (Pick #)</font> | ||
|- | |- | ||
| 2. | | 2. C-His(E/P)/ 25 ng || | ||
|} | |} | ||
Line 109: | Line 65: | ||
| || 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) | |||
* Concentrations: (1) 260 = , 260/280 = , '''ng/μL = '''; (2) 260 = , 260/280 = , '''ng/μL = '''; | |||
* Digest 10 μL of each with X/S for Gibson cloning (Brady and Vi) | |||
{| | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
| DNA ( | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>3. pSB1A3 = size<br>4. BB2 = size<br> | |||
| rowspan="7" | [[Image:KAH121112_gel2.png|400px|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 || | | dH<sub>2</sub>O || 15.0 | ||
|- | |- | ||
| || | | || 15 μL --> 37°C/ ~30 min. | ||
|} | |} | ||
Revision as of 16:29, 11 December 2012
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
12/11/12
Assemblies
Minipreps
|