User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20: Difference between revisions
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== | ==03/20/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Oligo annealing, round 2 | ||
* | * Assembly: annealed part into MV2 | ||
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---- | ---- | ||
'''Assemblies''' | '''Assemblies''' | ||
* 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. | |||
'''Oligo annealing''' | * '''Oligo annealing''' | ||
* Redo oligo annealing with 99.9 and 97.9°C starting temperatures... | |||
* Previous Thermal cycling (3/19/13) | |||
** 99.9, 10 min. | |||
** [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35 | |||
** 25°C, ∞ | |||
** Labeled tubes A1, A2; Stored on bench o/n | |||
* New Thermal cycling (3/20/13) | |||
** 99.9, 10 min. | |||
** [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36 | |||
** 25°C, ∞ | |||
** Labeled tubes A3, A4 | |||
* | * '''Digest''' (Fermentas FD) | ||
** MV2, XbaI only | ** MV2, XbaI only | ||
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| dH<sub>2</sub>O || 5.0 | | dH<sub>2</sub>O || 5.0 | ||
|- | |- | ||
| || 30 μL --> 37°C/ ~ | | || 30 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
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* Dephosphorylation (Roche) | * '''Dephosphorylation''' (Roche) | ||
{| {{table}} cellspacing="3" <!-- Dephos table --> | {| {{table}} cellspacing="3" <!-- Dephos table --> | ||
|- | |- | ||
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| 10x buffer d.p. || 2.0 | | 10x buffer d.p. || 2.0 | ||
|- | |- | ||
| phosphatase || 1.0 | | rapid alk. phosphatase || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 10.8 | | dH<sub>2</sub>O || 10.8 | ||
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{| {{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> 03/21/13</font> | ||
|- | |||
| 1. insert(Oligo S/S)/size, ## ng + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font> | |||
|- | |||
| 2. insert(Oligo S/S)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font> | |||
|- | |- | ||
| | | 3. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font> | ||
|- | |- | ||
| | | 4. vector(c/d)/ ## ng || | ||
|} | |} | ||
Revision as of 13:42, 20 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> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
03/20/13
Assemblies
Oligo annealing
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