User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06: Difference between revisions
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== | ==01/06/13== | ||
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* | * Gibson Assembly: pick colonies & check via colony PCR | ||
* | * Media: Make LB agar +amp | ||
* Gibson Assembly: redo transformation with more DNA | |||
---- | ---- | ||
''' | '''Gibson Assembly results'''<br> | ||
* | * Plates from [http://openwetware.org/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/05 01/05/2013] | ||
* hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2) | |||
* hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4) | |||
* hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6) | |||
* hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8) | |||
* | |||
* Pick and streak colonies from plates 3, 5, 7 | |||
* Use same pipette tip for colony PCR | |||
{| {{table}} cellspacing="3" <!-- Dephos table --> | {| {{table}} cellspacing="3" <!-- Dephos table --> | ||
|- | |- | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| bgcolor=#cfcfcf | 5x mix | |||
| rowspan="7" | [[Image:KAH010613_gel1.jpg|270px|PCR gel]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | |||
|- | |- | ||
| | | Colony || --- || --- | ||
|- | |- | ||
| | | 10 μM primer BL9 || 1.0 || 5.0 | ||
|- | |- | ||
| | | 10 μM primer BL10 || 1.0 || 5.0 | ||
|- | |- | ||
| | | 2x GoTaq || 12.5 || 62.5 | ||
|- | |- | ||
| | | dH<sub>2</sub>O || 10.5 || 52.5 | ||
|- | |||
| || 25.0 μL || | |||
|} | |} | ||
PCR | |||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C) | |||
---- | ---- | ||
''' | '''Transformation''' | ||
# | # hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson | ||
# | # V0120, 6.0 μL 1/4x Gibson | ||
# hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson | |||
# pSB1A3, 6.0 μL 1/4x Gibson | |||
* Add DNA to 50 μL DH5α-Turbo; ice/ 10 min. | |||
* Plate on 100 μg/mL amp. | |||
Revision as of 17:38, 6 January 2013
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01/06/13
Gibson Assembly results
PCR
Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)
Transformation
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