User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/12: Difference between revisions

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Line 67: Line 67:
| insert || 2.0 || ---
| insert || 2.0 || ---
|-
|-
| 10x FD buf || 2.0 || 18.0
| 10x FD buf || 2.0 || 10.0
|-
|-
| 10 mM DTT || 1.0 || 9.0
| 10 mM DTT || 1.0 || 5.0
|-
|-
| 10 mM ATP || 1.0 || 9.0
| 10 mM ATP || 1.0 || 5.0
|-
|-
| FastDigest BbsI/BpiI || 1.0 || 9.0
| FastDigest BbsI/BpiI || 1.0 || 5.0
|-
|-
| Roche T4 DNA ligase || 0.5 || 4.5
| Roche T4 DNA ligase || 0.5 || 2.5
|-
|-
| dH<sub>2</sub>O || ### || ---
| dH<sub>2</sub>O || ### || ---

Revision as of 14:41, 12 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/12/15

  • Cas-tone - streak Addgene plasmids for single colonies; design histone primers
  • Ryan - re-treat insert DNA (heat, slow anneal); redo dig/lig


Ryan - retry dig/lig

  • Idea: small inserts may have melted and re-annealed improperly during 80°C enzyme deactivation
  • Use remaining PCR reaction for another digest/phosphatase treatment.
  • Do all steps in thermal cycler


  • Digest & dephos PCR fragments
Reagent Volume
DNA (500 ng) 10.0 μL
10X buffer 2.0
EcoRI 1.0
XbaI 1.0
SAP (Roche) 1.0
dH2O 5.0
  20.0


  • LabNet OptiMax Thermocycler: AnOlig RD
    • 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, ∞


  • Dig/Lig reactions
    • 2:1 ratio calculation: ~150 bp insert / ~4000 bp vector * 2 * 100 = 3.75 ng insert
  1. pAub-PCR(E/S dp) + AubR/MRV
  2. pBja-PCR(E/S dp) + BjaR/MRV
  3. pBra-PCR(E/S dp) + BraR/MRV
  4. pRpa-PCR(E/S dp) + RpaR/MRV


Reagent Rxn Mix (x5)
100 ng Vector ### (up to 13.5) ---
insert 2.0 ---
10x FD buf 2.0 10.0
10 mM DTT 1.0 5.0
10 mM ATP 1.0 5.0
FastDigest BbsI/BpiI 1.0 5.0
Roche T4 DNA ligase 0.5 2.5
dH2O ### ---
  20.0

--> Pipette 5.5 μL master mix into each PCR tube
--> Add 2.0 μL insert into each tube
--> Add 100 ng vector DNA
--> Add x μL dH2O = 13.5 - vector volume
--> Mix by flicking

LabNet OptiMax Thermocycler: BbsI Dig/Lig

  • 6x [37°C, 5 min; 23°C, 5 min]
  • 4°C, ∞


Transformation(s)

  • Thaw 2 tubes chem competent DH5α-turbo on ice
  • Transfer 10.0 μL each ligation (1/2 rxn.) to fresh sterile 0.5 mL tube
  • Add 50 μL DH5α-turbo; pipette up-and-down 3x GENTLY
  • Incubate 5 min. on ice
  • Plate on 100 μg/mL amp


RESULTS (5/13/15)



Minipreps

  • Check with E/P digests
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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