User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/21: Difference between revisions
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(Autocreate 2013/03/21 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning) |
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==mm/dd/yy== | ==mm/dd/yy== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Assemblies: retry MV9 | ||
* | * T4 ligase quality check: Behzad's tube | ||
---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
* Try again, but with the old boil and cool method | |||
* | * '''Oligo annealing''' | ||
** | * See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/19 03/19/13] for details about the oligos and reaction set-up | ||
** Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C). | |||
** Float the oligo mixtures in the boiling water for 10 min. If there is condensation, carefully & quickly remove the tube and shoe the liquid down to the bottom and put the tubes back into the water. | |||
** Turn off the heat source and allow the water bath to slowly cool to room temperature (25°C) for several hours. | |||
* '''Digest''' (Fermentas FD) - MV2 vector, [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13] | |||
* '''Measure conc.''' - [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13] | |||
* '''Dephosphorylation''' (Roche) - MV2 XbaI-cut vector, [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13] | |||
* '''Ligations''' | |||
* | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
{| {{table}} cellspacing="3" <!-- | |- bgcolor=#cfcfcf | ||
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font> | |||
|- | |- | ||
| | | 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font> | ||
| | |||
|- | |- | ||
| | | 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font> | ||
|- | |- | ||
| | | 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font> | ||
|- | |- | ||
| | | 4. MV9(X dp)/ 25 ng || | ||
| | |||
|- | |- | ||
| | | 5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase || <font color="blue">...</font> | ||
|- | |- | ||
| | | 6. MV9(X no dp)/ 25 ng || | ||
|} | |} | ||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || 3 || 4 || 9 || 10 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.5 || 1.0 || 2.0 || --- || --- || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 1.0 || 1.0 || 1.0 || 1.0 || 0.3 || 0.3 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || --- | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 2.5 || 2.0 || 1.0 || 3.0 || 3.7 || 4.7 | ||
|- | |- | ||
| || | | || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL | ||
|} | |} | ||
* 10 min./ room temp | |||
* Add 30 μL DH5α; 5 min/ ice | |||
* Plate on 100 μg/mL amp; incubate at 37°C | |||
<!-- ##### 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. ##### --> |
Revision as of 19:48, 21 March 2013
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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
mm/dd/yy
Assemblies
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