User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09: Difference between revisions
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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Golden Gate assembly: hPCD + BL01 + pSB1A3 | * Golden Gate assembly: hPCD + BL01 + pSB1A3 | ||
* Violacein: plates from Tabor; incubate o/n | |||
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* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites | * Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites | ||
Dilutions | * Dilutions | ||
{| {{table}} border="1" cellspacing="3" <!-- | {| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | DNA | | bgcolor=#cfcfcf | DNA | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | length (bp) | ||
| bgcolor=#cfcfcf | ng/μL | |||
| bgcolor=#cfcfcf | Vol. | | bgcolor=#cfcfcf | Vol. | ||
| bgcolor=#cfcfcf | + dH<sub>2</sub>O | | bgcolor=#cfcfcf | + dH<sub>2</sub>O | ||
|- | |- | ||
| gg2 || 67.7 || | | gg2 pSB1A3 || 2000 || 67.7 || 7.7 || 12.3 | ||
|- | |- | ||
| gg3 || 22.5 || | | gg3 hPCD || 186 || 22.5 || 2.1 || 17.8 | ||
|- | |- | ||
| gg4 || 55.9 || | | gg4 BL01 || 2520 || 55.9 || 11.7 || 8.3 | ||
|} | |} | ||
* Golden Gate Reactions | |||
# hPCD + BL01 + pSB1A3 | |||
# pSB1A3 | |||
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table --> | |||
|- valign="top" | |||
| Reagent || 1 || 2 | |||
|- | |||
| gg2 pSB1A3 || 1.0 || 1.0 | |||
|- | |||
| gg3 hPCD || 1.0 || --- | |||
|- | |||
| gg4 BL01 || 1.0 || --- | |||
|- | |||
| 10x ligase buffer || 1.0 || 1.0 | |||
|- | |||
| NEB T4 lgase || 0.25 || 0.25 | |||
|- | |||
| BsmBI || 0.5 || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 5.25 || 7.25 | |||
|- | |||
| || 10.0 || 10.0 | |||
|} | |||
Thermal cycler | |||
* [45°C, 2 min.; 16°C, 5 min.] x25 | |||
* 60°C, 20 min. | |||
* 80°C, 20 min. | |||
Revision as of 17:57, 10 January 2013
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01/09/13
Golden Gate Assembly
Thermal cycler
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