User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/06: Difference between revisions

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Part 1: Prep the Oligo Bridges
Part 1: Prep the Oligo Bridges
* Note: Final conc. in LCR rxn. is 30 nM
* Note: Final conc. in LCR rxn. is 30 nM each
* Bring the IDT oligo pellet to 100μM with dH<sub>2</sub>O.
* Bring the IDT oligo pellet to 100μM with dH<sub>2</sub>O.
** x nmoles oligo (on label) * 10 = y μL H<sub>2</sub>O to add
** x nmoles oligo (on label) * 10 = y μL H<sub>2</sub>O to add
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Part 2: Prep the DNA fragments
Part 2: Prep the DNA fragments
* Note: Final conc. in LCR rxn is 30 nM
* Note: Final conc. in LCR rxn is 3 nM each
* Option 1 - PCR (scarless)
* Option 1 - PCR (scarless)
** Use primers with 5' phosphates to amplify the fragment(s) of interest. Error-free PCR is highly recommended for large fragments.  
** Use primers with 5' phosphates to amplify the fragment(s) of interest. Error-free PCR is highly recommended for large fragments.  
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LCR Calculations
Part 3: LCR Calculations
* 3 nM dsDNA = length * ...
{|
|-
| DNA | Type | Target conc. |
 
 
1:1 vector to insert
1:1 vector to insert
BL01: (2520bp/5197bp)(1)(50ng)=24.24ng
BL01: (2520bp/5197bp)(1)(50ng)=24.24ng

Revision as of 13:41, 3 April 2015

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04/06/15

  • LCR - continued
  • Order new oligos - MV10 (5' phos)


LCR Continued


Part 1: Prep the Oligo Bridges

  • Note: Final conc. in LCR rxn. is 30 nM each
  • Bring the IDT oligo pellet to 100μM with dH2O.
    • x nmoles oligo (on label) * 10 = y μL H2O to add
  • Make a 300 nM working solution (final volume = 1000 μL) in a new tube.
    • 3 μL of 100μM oligo stock + 997 μL dH2O = 1000 μL
  • Note: Final conc. in LCR rxn. is 30 nM


Part 2: Prep the DNA fragments

  • Note: Final conc. in LCR rxn is 3 nM each
  • Option 1 - PCR (scarless)
    • Use primers with 5' phosphates to amplify the fragment(s) of interest. Error-free PCR is highly recommended for large fragments.
    • Purify the product with a kit of choice (e.g. Sigma PCR clean-up)
  • Option 2 - Restriction digest (will produce scars)
    • Ensure that the digest does not produce overhangs that will produce unwanted ligations (i.e. five fragments that are all cut with EcoRI)
    • The oligo bridge should include the sequences from the overhangs that will end up in the final assembled plasmid
    • The easiest way to design oligo bridges in this case is to build the final plasmid in silico, then select bridge oligos that span the junction, including the assembly scar


Part 3: LCR Calculations

  • 3 nM dsDNA = length * ...
Type | Target conc. |


1:1 vector to insert BL01: (2520bp/5197bp)(1)(50ng)=24.24ng 24.24ng(1μL/27ng)=0.897μL XbaI/SpeI cut BL01 BL05: (2520bp/5197bp)(1)(50ng)=24.24ng 24.24ng(1μL/30ng)=0.808μL XbaI/SpeI cut BL05 50ngCMV/MV9(1μL/59ng)=0.847μL XbaI cut CMV/MV9


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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