User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/22: Difference between revisions

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| 2. 5'p MV10 PCR || --- || --- || ---
| 2. 5'p MV10 PCR || --- || --- || ---
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Dilute the purified dsDNA to 30 fmol/μL (30 nM)
* Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
* Add x to dH<sub>2</sub>O, final vol. = 50 μL
* Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction




Revision as of 15:59, 22 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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04/22/15

  • Ryan - streak plates & liquid cultures (see 4/21/15)
  • Rene - minipreps: g53-2, g54
  • LCR Development - assembly attempt #1


LCR - assembly attempt #1


Column-purify PCR fragments

  • Use Qiagen PCR Clean-up, elute and back-elute w/ 30 μL elution buffer
  1. Gal4DB-mCh (4/22/15)
  2. ATF2 (4/22/15)
  3. MV10 (4/17/15)


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 5'p Gal4DB-mCh PCR --- --- ---
2. 5'p ATF2 PCR --- --- ---
2. 5'p MV10 PCR --- --- ---


Dilute the purified dsDNA to 30 fmol/μL (30 nM)

  • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
  • Add x to dH2O, final vol. = 50 μL
  • Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction


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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