User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09: Difference between revisions
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== | ==01/09/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Golden Gate assembly: hPCD + BL01 + pSB1A3 | ||
* | * Violacein: plates from Tabor; incubate o/n | ||
---- | ---- | ||
''' | '''Golden Gate Assembly'''<br> | ||
* | * Use purified, measured PCR products from 1/05/13 | ||
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites | |||
{| {{table}} border="1" cellspacing="3" <!-- | * Dilutions | ||
{| {{table}} border="1" cellspacing="3" <!-- Dilution table --> | |||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | DNA | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | length (bp) | ||
| | | bgcolor=#cfcfcf | ng/μL | ||
| | | bgcolor=#cfcfcf | Vol. | ||
| bgcolor=#cfcfcf | + dH<sub>2</sub>O | |||
|- | |- | ||
| | | gg2 pSB1A3 || 2000 || 67.7 || 7.7 || 12.3 | ||
|- | |- | ||
| | | gg3 hPCD || 186 || 22.5 || 2.1 || 17.8 | ||
|- | |- | ||
| | | 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 --> | |||
{| {{table}} cellspacing="3" <!-- | |||
|- valign="top" | |- valign="top" | ||
| Reagent || 1 || 2 | |||
| | |||
| | |||
| | |||
|- | |- | ||
| 1. | | 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 | | 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
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> | |||||||||||||||||||||||||||||||||||||||||||||||
01/09/13
Golden Gate Assembly
Thermal cycler
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