User:Karmella Haynes/Notebook/BioBrick cloning/2015/03/26

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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



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