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

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
1	1 kb Ladder (4 μL)	-
2	folded 6 hb (4 μL)	2 μL
3	negative control: no scaffold (4 μL)	2 μL
4	negative 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 μL	3%	30 μL	20 μL	110 μL	200 μL
6hb: 40 μL	4%	40 μL	20 μL	100 μL	200 μL
6hb: 40 μL	5%	50 μL	20 μL	90 μL	200 μL
6hb: 40 μL	10%	100 μL	20 μL	40 μL	200 μL
6hb: 40 μL	14%	140 μL	20 μL	0 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
1	1 kb Ladder (4 μL)	-
2	untreated 6hb (10 μL)	2 μL
3	3% supernatant (10 μL)	2 μL
4	3% pellet (10 μL)	2 μL
5	4% supernatant (10 μL)	2 μL
6	4% pellet (10 μL)	2 μL
7	5% supernatant (10 μL)	2 μL
8	5% pellet (10 μL)	2 μL
9	10% supernatant (10 μL)	2 μL
10	10% pellet (10 μL)	2 μL
11	14% supernatant (10 μL)	2 μL
12	14% pellet (10 μL)	2 μL