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, 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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| || 10 mL | | || 10 mL | ||
|} | |} | ||
'''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
Procedure
Assemblies
Oligo annealing
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