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

Karmella's BioBrick Cloning

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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