User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/11: Difference between revisions

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* Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong)
* Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong)
* Cas-tone - PCR-amplify CMV
* Cas-tone - PCR-amplify CMV
* Ryan - minipreps & digests




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* Conclusions
** Success!




Revision as of 19:41, 11 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/11/15

  • Cancer PcTF - cultures for KAH87_MV10 clone #1 from streak library (clone #3 is wrong)
  • Cas-tone - PCR-amplify CMV
  • Ryan - minipreps & digests


Cas-tone Project

  • STAGE 1 - pcDNA-dCas9-VP64 vector re-design
    • Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
    • 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
    • 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
    • Order primers
  • STAGE 3 - Cas-fusion
    • Design primers for H2A, H2B, H3, H4, and tails
    • Insert Kozak-histone parts (X/N) into dCas9 (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA
  • Application
    • Cas and gRNA plasmids will be co-transformed
    • Analyze Cas protein via Western blot


Knock-out FLAG from N-terminus

  • PCR-amplify promoters
    • 2x 50 μL rxns
    • Template = CMV (KAH
    • f primer: XbaI-CMV f1 - 5'-CCTTTCTAGAGTTGACATTGATTATTGGCTAG
    • r primer: SacII-NS-CMV r1 - 5'-CATTCCGCGGGCGGCCGCTACTAGTGAGCTCTGC


Reagent Vol Mix (x2) Expected:
1. CMV = 588 		Hover name
5 μL/lane; 1% agarose; Ladder
plasmid DNA 0.5 1.0
10 μM XbaI-CMV f1 1.0 2.0
10 SacII-NS-CMV r1 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 45.0
  50.0 μL ---

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 55°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞


  • Conclusions
    • Success!



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