User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05: Difference between revisions
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* Golden gate: PCR parts, complete protocol for Brady's construct | * Golden gate: PCR parts, complete protocol for Brady's construct | ||
* Gibson: PCR parts, complete protocol for Brady's construct | * Gibson: PCR parts, complete protocol for Brady's construct | ||
---- | |||
'''Gibson Assembly'''<br> | |||
# hPCD + BL01 + V0120 | |||
# hPCD + BL01 + pSB1A3 | |||
'''PCR''' | |||
# V0120, BBS F / BBP R | |||
# pSB1A3, gg_BBS F / gg_BBP R | |||
# hPCD, gg_BBP-hPCD F / gg_hPCD-BL01 R | |||
# BL01, gg_BL01 F / gg_BL01-BBS R | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
| rowspan="7" | <u>Expected:</u><br>1. V0120 = size<br>2. pSB1A3 = size<br>3. hPCD = size<br>4. BL01 = size | |||
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] --> | |||
|- | |||
| DNA(plasmid) || 0.5 μL | |||
|- | |||
| 10 μM primer 1 || 1.0 | |||
|- | |||
| 10 μM primer 2 || 1.0 | |||
|- | |||
| 2x GoTaq mix || 25.0 | |||
|- | |||
| dH<sub>2</sub>O || ## | |||
|- | |||
| || 50 μL | |||
|} | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
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# hPCD + BL01 + pSB1A3 | # hPCD + BL01 + pSB1A3 | ||
* Check the sequences for BsmBI sites | * Check the sequences for BsmBI sites! | ||
'''PCR''' | '''PCR''' | ||
# V0120, | # V0120, gg_BBS F / gg_BBP R | ||
# pSB1A3, | # pSB1A3, gg_BBS F / gg_BBP R | ||
# hPCD, gg_BBP-hPCD F / gg_hPCD R | # hPCD, gg_BBP-hPCD F / gg_hPCD-BL01 R | ||
# BL01, gg_BL01 F / gg_BL01-BBS R | # BL01, gg_BL01 F / gg_BL01-BBS R | ||
Revision as of 11:35, 5 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> | ||||||||||||||||||||||||||||||||
01/05/13
Gibson Assembly
Golden Gate Assembly
PCR
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