User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions
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* LCR Development - PCR MV10 | * LCR Development - PCR MV10 backbone | ||
* gRNA cloning (Rene) | * gRNA cloning (Rene) | ||
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| || 50.0 || 50.0 | | || 50.0 || 50.0 | ||
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<font color=red>Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run</font> | |||
Program: Phusion (block B) | Program: Phusion (block B) | ||
* 98°C, 3 min | * 98°C, 3 min | ||
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, | * 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | ||
* 72°C, 10 min | * 72°C, 10 min | ||
* 4°C ∞ | * 4°C ∞ | ||
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* Plate on 100 μg/mL amp | * Plate on 100 μg/mL amp | ||
** Split ligations in 1/2, do rapid protocl for 1 set, long protocol for other set | ** Split ligations in 1/2, do rapid protocl for 1 set, long protocol for other set | ||
RESULTS (4/17/15) | |||
* Success! Paltes 1-3, 5, 6 had ~5 colonies. Plate 4 had just one. | |||
* Pick two colonies from each (except plate 4, pick 1) | |||
* Make streak plate & set up 5 mL cultures | |||
Revision as of 12:08, 17 April 2015
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04/16/15
LCR Development
Measure conc.
Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run Program: Phusion (block B)
Conclusion
gRNA cloning - Rene
Phosphorylate and anneal oligo pairs
LabNet OptiMax Thermocycler: BbsI Dig/Lig
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