User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/08

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10/09/15

  • Cas-tone- H3 inserts into MV12 (MV11-GFP)


Cas-tone Project

  • STAGE 1 - pcDNA-dCas9-VP64 vector re-design
    • DONE - Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo
    • 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
    • DONE - Order primers (annotated in Benchling)
    • DONE PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer)
  • STAGE 3 - Cas-fusion
    • Insert Kozak-histone parts (X/N) into dCas9 plasmid "MV12" (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Assemblies

  1. H3-1-40_MV11: H3-1-40/(X/N)/129 + MV12/(S/N)/9506
  2. H3-1-60_MV11: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV11: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV11: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV11: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
    • SpeI/NotI
Reagent Volume
DNA (plasmid) 15.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O 10.0
  30 μL --> 37°C/ ~15 min.
  • Gel purification
    • Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.


  • DpnI digest & clean-up of PCR products
    • Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
    • Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
    • Elute & back-elute with 30 μL of elution solution.


  • Measure [DNA] for vector and inserts
Sample OD260 260/280 ng/μL
1. MV11 (S/N) 0.03 1.879 13.5
2. H3-1-40 PCR 0.03 1.879 29.8
3. H3-1-60 PCR 0.04 1.974 39.7
4. H3-1-80 PCR 0.047 1.865 46.8
5. H3-1-116 PCR 0.06 1.887 59.57
6. H3-1-136 PCR 0.062 1.932 62.0


  • Digest & dephos inserts
Reagent Volume
DNA (500 ng) up to 15.0 μL
10X FD buffer 2.0
FD EcoRI 1.0
FD XbaI 1.0
Roche SAP 1.0
dH2O x μL
  20.0

Thermal cycler: LabNet OptiMax "AnOlig shrt"

  • 37°C, 10 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞
  • Final concentration = 25 ng/μL.


  • Ligations
    • > 2:1 ratio calculations...
    • 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
    • 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 3.5 3.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 0.5
  10.0 μL 10.0 μL

RESULTS (5/22/15)

  • Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony
  • Pick 2 colonies from each plate for 5 mL cultures and streaks



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