User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions
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'''Reaction set-up''' | |||
* Use 0.2 - 0.5 pmol, per NEB's suggestion | |||
* pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000 | |||
* Total volume of DNA = 5.0 μL max | |||
1. hPCD + BL01 + V0120 | |||
* vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL) | |||
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL | |||
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL | |||
2. hPCD + BL01 + V0120 | |||
* vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL | |||
* 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL) | |||
* 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL | |||
Gibson reactions: | |||
# hPCD + BL01 + V0120 | |||
# V0120 | |||
# hPCD + BL01 + pSB1A3 | |||
# pSB1A3 | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| 1 || 2 || 3 || 4 | |||
|- | |||
| Vector || 1.4 || 1.4 || 1.3 || 1.3 | |||
|- | |||
| Ins 1 || 0.7 || --- || 0.8 || --- | |||
|- | |||
| Ins 2 || 2.9 || --- || 2.9 || --- | |||
|- | |||
| dH<sub>2</sub>O || --- || 3.6 || --- || 3.7 | |||
|- | |||
| || 5.0 μL | |||
|} | |||
* Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL | |||
* Thermal cycler: 50°C,60 min.; 4°C, ∞ | |||
* Transform | |||
Revision as of 17:02, 5 January 2013
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01/05/13
Gibson Assembly
Measure DNA Concentrations
1. hPCD + BL01 + V0120
2. hPCD + BL01 + V0120
Golden Gate Assembly
PCR
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