User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/26: Difference between revisions
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** PCR-amplify HPK-CFP gene (DBN006) with new/ improved primers | ** PCR-amplify HPK-CFP gene (DBN006) with new/ improved primers | ||
** DBN006: https://benchling.com/s/XoQEXLOz/edit | ** DBN006: https://benchling.com/s/XoQEXLOz/edit | ||
** Primers: P072, P073 | |||
** Use Life Tech/ Thermo [https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html Tm calculator] to calculate annealing temperature | ** Use Life Tech/ Thermo [https://www.lifetechnologies.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html Tm calculator] to calculate annealing temperature | ||
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| bgcolor=#cfcfcf | Rxn1,2 | | bgcolor=#cfcfcf | Rxn1,2 | ||
| rowspan="9" | Expected:<br>1,2. DBN006 = 1972 | | rowspan="9" | Expected:<br>1,2. DBN006 = 1972 | ||
| rowspan="9" | [[Image: | | rowspan="9" | [[Image:KAH052615_gel1.jpg|150px|Hover name]]<br>5 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| Template || 0.2 | | Template || 0.2 | ||
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* 4°C ∞ | * 4°C ∞ | ||
CONCLUSION | |||
* Non-specific amplification. Need to gel purify. | |||
* Use 30 uL of each per lane for gel purification. | |||
* | |||
** Use | * Gel purification | ||
** Use Sigma kit | |||
** Elute & back-elute w/ 25 μL elution sln. | ** Elute & back-elute w/ 25 μL elution sln. | ||
* Digest & dephos | * Digest & dephos | ||
** PCR, Clip & Clone protocol; digest w/ BsaI | ** PCR, Clip & Clone protocol; digest w/ BsaI; omit DpnI | ||
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| bgcolor=#cfcfcf | Rxn1-24 | | bgcolor=#cfcfcf | Rxn1-24 | ||
| bgcolor=#cfcfcf | Mix (x25) | | bgcolor=#cfcfcf | Mix (x25) | ||
| rowspan="7" | Expected:<br>1 | | rowspan="7" | Expected:<br>1-14. PCR insert/ pJET = ~600 bp | ||
| rowspan="7" | [[Image: | | rowspan="7" | [[Image:KAH052615_gel3.jpg|250px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] | ||
|- | |- | ||
| Template (culture) || 0.5 || --- | | Template (culture) || 0.5 || --- | ||
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|} | |} | ||
Labnet: "GoTaq" | |||
* 98°C, 3 min | * 98°C, 3 min | ||
* | * 35x[95°C, 10 sec; 60°C, 10 sec; 72°C, 20 sec] | ||
* 72°C, | * 72°C, 3 min | ||
* 4°C ∞ | * 4°C ∞ | ||
CONCLUSIONS | |||
* Success with dirty PCR | |||
* Continue with remainder of dirty PCR & possible SYBR PCR tomorrow | |||
Revision as of 17:49, 26 May 2015
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05/26/15
Ben - luc replacement donor plasmid
Bio-Rad: "Phusion" (block #)
CONCLUSION
Rene - CRISPR PCR library PCR trial
1-12. Luc14 g034 - Lu34_AA01 - Lu34_AA12
Labnet: "GoTaq"
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