IGEM:Harvard/2006/vlau/Notebook/2006-8-2

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Folding Rxn

1. Goal: 6 samples of 100 μL 6 hb

2. Reaction mixture:

      50μL p7308 scaffold
      40μL oligos
      10μL 10x folding buffer (500mM HEPES ph 7.5, 500mM NaCl, 100mM MgCl2)
      neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water

3. Image (2% agarose gel + 11 mM MgCl2)

Lane Contents Loading Dye
11 kb Ladder (4 μL)-
2folded 6 hb (4 μL)2 μL
3negative control: no scaffold (4 μL)2 μL
4negative control: no oligos (4 μL)2 μL

PEG Precipitation

1. Goal: repeat titration of PEG precipitation conditions for 2 samples of 6hb

2. Protocol

Sample % PEG 20% PEG Volume 5 M NaCl Volume dH2O Total Volume
6hb: 40 μL3%30 μL20 μL110 μL200 μL
6hb: 40 μL4%40 μL20 μL100 μL200 μL
6hb: 40 μL5%50 μL20 μL90 μL200 μL
6hb: 40 μL10%100 μL20 μL40 μL200 μL
6hb: 40 μL14%140 μL20 μL0 μL200 μL


     - incubated on ice for 15 min.
     - spun @ 16k rcf for 10 min.
     - pipetted supernatant into separate tube
     - pellet resuspended in 1x folding buffer volume equal to supernatant
     - ran equivalent volumes of pellet and supernatant on 2% agarose gel (+11 mM MgCl2)

3. Gel Analysis

Lane Contents Loading Dye
11 kb Ladder (4 μL)-
2untreated 6hb (10 μL)2 μL
33% supernatant (10 μL)2 μL
43% pellet (10 μL)2 μL
54% supernatant (10 μL)2 μL
64% pellet (10 μL)2 μL
75% supernatant (10 μL)2 μL
85% pellet (10 μL)2 μL
910% supernatant (10 μL)2 μL
1010% pellet (10 μL)2 μL
1114% supernatant (10 μL)2 μL
1214% pellet (10 μL)2 μL
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