User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/11

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Karmella's BioBrick Cloning 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!
    • Proceed to PCR purification (PCR Clip & Clone protocol)


  • PCR clean-up
    • Qiagen PCR Purification kit
    • Elute & back-elute w/ 30 μL elution buffer


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. XbaI-CMV-SpeI-NotI-SacII PCR 0.186 1.868 186.4



Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • Cut w/ E/KpnI
    • See my version of MRV map: https://benchling.com/s/oJ73uatK/edit
    • Note: BbsI drop-in of promoter knocks out KpnI site (54); no E, X, or S sites are restored by promoter insert
    • Note: EcoRI (1025) and XbaI (1040) were maintained after Regulator insertion
  1. pAub-AubR/MRV
  2. pBja-BjaR/MRV
  3. pBra-BraR/MRV
  4. pRpa-RpaR/MRV


Reagent Volume Expected:
1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = 4191
7,8. pRpa/RpaR/MRV = 4116
9. Empty MRV = 2230, 971

np pro. Reg/MRV = ~4000, 971
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
KpnI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pAub-AubR/MRV-1 0.152 1.930 152.3
2. pAub-AubR/MRV-2 0.079 1.992 78.6
3. pBja-BjaR/MRV-1 0.093 1.989 92.7
4. pBja-BjaR/MRV-2 0.043 1.934 43.1
5. pBra-BraR/MRV-1 0.070 2.021 69.9
6. pBra-BraR/MRV-2 0.030 1.946 29.8
7. pRpa-RpaR/MRV-1 0.093 1.956 92.6
8. pRpa-RpaR/MRV-2 0.033 2.000 32.7


Conclusions

  • Clones 1, 7, 8 look like empty Regulator-MRV vectors



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