User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20: Difference between revisions
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* 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. This way, the vector can be customized to carry different promoters. | ||
** Insertion of the ds oligo should destroy the vector's XbaI site | |||
** Will need to check inserts for copy number and orientation | |||
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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. | | 1. A1 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | ||
|- | |- | ||
| 2. | | 2. A1 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | ||
|- | |- | ||
| 3. | | 3. A1 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | ||
|- | |- | ||
| 4. | | 4. MV9(X dp)/ 25 ng || | ||
|- | |||
| 5. A3 (ds oligo S/S)/size, ''0.5 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | |||
|- | |||
| 6. A3 (ds oligo S/S)/size, ''1.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | |||
|- | |||
| 7. A3 (ds oligo S/S)/size, ''2.0 μL'' + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | |||
|- | |||
| 8. MV9(X dp)/ 25 ng || | |||
|} | |} | ||
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| || 1 || 2 || | | || 1 || 2 || | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.5 || --- || | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 1.0 || 1.0 || | ||
|- | |- | ||
| 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 || | ||
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| dH<sub>2</sub>O || ### || ### || | | dH<sub>2</sub>O || ### || ### || | ||
|- | |- | ||
| || | | || 10 μL || 10 μL || | ||
|} | |} | ||
Revision as of 13:49, 20 March 2013
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03/20/13
Assemblies
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