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

Folding Rxn

1. goal: 8 samples of 40 μL 6 hb 2. reaction mixture:

      9μL p7308 scaffold
      4μL oligos
      4μL 10x folding buffer (500mM HEPES ph 7.5, 500mM NaCl, 100mM MgCl2)
      23μL dH2O
      neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water

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
8/1 6hb: 40 μL	3%	30 μL	20 μL	110 μL	200 μL
8/1 6hb: 40 μL	4%	40 μL	20 μL	100 μL	200 μL
8/1 6hb: 40 μL	5%	50 μL	20 μL	90 μL	200 μL
old 6hb: 40 μL	3%	30 μL	20 μL	110 μL	200 μL
old 6hb: 40 μL	4%	40 μL	20 μL	100 μL	200 μL
old 6hb: 40 μL	5%	50 μL	20 μL	90 μL	200 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
8/1 6hb: 40 μL	3%	30 μL	20 μL	110 μL	200 μL
8/1 6hb: 40 μL	4%	40 μL	20 μL	100 μL	200 μL
8/1 6hb: 40 μL	5%	50 μL	20 μL	90 μL	200 μL
old 6hb: 40 μL	3%	30 μL	20 μL	110 μL	200 μL
old 6hb: 40 μL	4%	40 μL	20 μL	100 μL	200 μL
old 6hb: 40 μL	5%	50 μL	20 μL	90 μL	200 μL