User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/18: Difference between revisions
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** '''DONE''' - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII | ** '''DONE''' - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII | ||
* STAGE 2 - Histone parts | * STAGE 2 - Histone parts | ||
** '''DONE''' - Order primers (annotated in Benchling) | |||
** PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail | ** PCR-clone histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will introduce unnatural amino acids onto conserved histone N-terminal tail | ||
* STAGE 3 - Cas-fusion | * STAGE 3 - Cas-fusion | ||
** Insert Kozak-histone parts (X/N) into dCas9 (S/N) | ** Insert Kozak-histone parts (X/N) into dCas9 (S/N) |
Revision as of 13:05, 19 May 2015
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05/18/15
Ben - assembly: HPK-CFP into homology arms
Program: Phusion (block B)
Conclusion
Cas-tone Project
Assembly
--> 37°C/ ~30 min.
LabNet OptiMax Thermocycler: AnOlig RD
Dilute the product(s) 1:250
Rene - PCR cloning trial 3
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