User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20: Difference between revisions
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'''Assemblies''' | '''Assemblies''' | ||
* New strategy for mammalian vector: | * New strategy for mammalian vector: | ||
** Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter. This way, the vector can be customized to carry different promoters. | ** Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter (MV2, puro). This way, the vector can be customized to carry different promoters. | ||
** Insertion of the ds oligo should destroy the vector's XbaI site | ** Insertion of the ds oligo should destroy the vector's XbaI site | ||
** Will need to check inserts for copy number and orientation | ** Will need to check inserts for copy number and orientation | ||
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* '''Oligo annealing''' | * '''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 | |||
* Redo oligo annealing with 99.9 and 97.9°C starting temperatures... | * Redo oligo annealing with 99.9 and 97.9°C starting temperatures... | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| rowspan="7" | | | rowspan="7" | [[Image:KAH032013_gel1.jpg|100px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| DNA (plasmid) || 20.0 μL | | DNA (plasmid) || 20.0 μL | ||
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|} | |} | ||
* Gel purify using the Zymo DNA Gel Recovery kit. | |||
* Elute with 20 μL dH<sub>2</sub>O | |||
* Measure conc.' | |||
* '''Measure conc.''' | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| | | MV2 (XbaI) || 6.2 μL (500 ng) | ||
|- | |- | ||
| 10x buffer d.p. || 2.0 | | 10x buffer d.p. || 2.0 | ||
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font> | | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font> | ||
|- | |- | ||
| 1. A1 (ds oligo S/S)/ | | 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 2. A1 (ds oligo S/S)/ | | 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 3. A1 (ds oligo S/S)/ | | 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 4. MV9(X dp)/ 25 ng || | | 4. MV9(X dp)/ 25 ng || | ||
|- | |- | ||
| 5. A3 (ds oligo S/S)/ | | 5. A3 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 6. A3 (ds oligo S/S)/ | | 6. A3 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 7. A3 (ds oligo S/S)/ | | 7. A3 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 failed</font> | ||
|- | |- | ||
| 8. MV9(X dp)/ 25 ng || | | 8. MV9(X dp)/ 25 ng || | ||
|- | |||
| 9. MV9(X no dp)/ 25 ng + T4 ligase || <font color="blue">Re-ligation successful, ~40 colonies</font> | |||
|- | |||
| 10. MV9(X no dp)/ 25 ng || | |||
|} | |} | ||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 || 10 | ||
|- | |- | ||
| Insert DNA || 0.5 || --- || | | Insert DNA || 0.5 || 1.0 || 2.0 || --- || 0.5 || 1.0 || 2.0 || --- || --- || --- | ||
|- | |- | ||
| Vector DNA || 1.0 || 1.0 || | | Vector DNA || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 0.3 || 0.3 | ||
|- | |- | ||
| 2x lgn buf (Roche) || 5.0 || 5.0 || | | 2x lgn buf (Roche) || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 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 || 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 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 | |||
---- | |||
3/21/13<br> | |||
Results for plates 9 and 10 | |||
[[Image:KAH032113_plates1.jpg|300px|Hover name]] | |||
Revision as of 14:50, 21 March 2013
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03/20/13
Assemblies
3/21/13 Results for plates 9 and 10
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