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

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| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (500 ng) || up to 15.0 μL
| DNA (500 ng) || 10.0 μL
|-
|-
| 10X buffer || 2.0
| 10X buffer || 2.0
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| SAP (Roche) || 1.0
| SAP (Roche) || 1.0
|-
|-
| dH<sub>2</sub>O || x μL
| dH<sub>2</sub>O || 5.0
|-
|-
| &nbsp; || 20.0
| &nbsp; || 20.0
|}
|}


Thermal cycler: LabNet


LabNet OptiMax Thermocycler: AnOlig RD
* LabNet OptiMax Thermocycler: AnOlig RD
37°C, 10 min
** 37°C, 10 min
95°C, 5 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]
** Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
25°C, ∞
** 25°C, ∞




Revision as of 14:36, 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



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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