User:Karmella Haynes/Notebook/BioBrick cloning/2015/03/26: Difference between revisions

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'''Make PEG solutions'''
'''Make PEG solutions'''
* Make TE Buffer, pH 8.0: 10 mM TRIS-HCl, 1 mM EDTA, pH 8.0
* Make TE Buffer, pH 8.0: 10 mM TRIS-HCl, 0.1 mM EDTA, pH 8.0
* Make 30 mM MgCl2, 50 mL (plus one extra): 1.5 mL 1M MgCl2 + 48.5 molecular bio grade H<sub>2</sub>O
* Make 30 mM MgCl2, 50 mL (plus one extra): 1.5 mL 1M MgCl2 + 48.5 molecular bio grade H<sub>2</sub>O
* Make 30% PEG, 50 mL: 1.5 g PEG 8000 + 50 mL 30 mM MgCL2
* Make 30% PEG, 50 mL: 1.5 g PEG 8000 + 50 mL 30 mM MgCL2
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| &nbsp; || 10 mL
| &nbsp; || 10 mL
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'''Procedure'''
* Make 6 samples of DNA ladder (1 kb Plus): 1 μL (500 ng) + 49 μL H<sub>2</sub>O
* Mix 50 μL of sample with 150 µL of TE
* Add 100 µL of PEG/MgCl2
* Vortex
* Centrifuge 15 min at 10,000 rcf at room temperature. Note: orient the tubes "hinge" side up so that the onvisible pellet will be located towards the hinge (at bottom of tube).
* Carefully transfer ALL supernatant to a new tube. Do not to disturb the pellet, which will be invisible
* Dissolve the pellet in 50 μL dH<sub>2</sub>O


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Revision as of 12:25, 26 March 2015

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03/26/15

  • PEG precipitation - optimization


PEG precipitation - optimization

  • See OWW protocol - Size selective DNA precipitation
  • In this procedure, PEG is diluted 3-fold in the final DNA mixture
  • Try these final PEG concentrations: 10%, 6%, 5%, 4%, 3%, 2%


Make PEG solutions

  • Make TE Buffer, pH 8.0: 10 mM TRIS-HCl, 0.1 mM EDTA, pH 8.0
  • Make 30 mM MgCl2, 50 mL (plus one extra): 1.5 mL 1M MgCl2 + 48.5 molecular bio grade H2O
  • Make 30% PEG, 50 mL: 1.5 g PEG 8000 + 50 mL 30 mM MgCL2
  • Use this to make other solutions
Reagent 18% PEG 15% PEG 12% PEG 9% PEG 6% PEG Expected:
1. BB 1 = size
2. BB2 = size
30% PEG 6.0 mL 5.0 mL 4.0 mL 3.0 mL 2.0 mL
30 mM MgCl2 4.0 mL 5.0 mL 6.0 mL 7.0 mL 8.0 mL
  10 mL

Procedure

  • Make 6 samples of DNA ladder (1 kb Plus): 1 μL (500 ng) + 49 μL H2O
  • Mix 50 μL of sample with 150 µL of TE
  • Add 100 µL of PEG/MgCl2
  • Vortex
  • Centrifuge 15 min at 10,000 rcf at room temperature. Note: orient the tubes "hinge" side up so that the onvisible pellet will be located towards the hinge (at bottom of tube).
  • Carefully transfer ALL supernatant to a new tube. Do not to disturb the pellet, which will be invisible
  • Dissolve the pellet in 50 μL dH2O

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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