IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-3

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Superconcentration Experiments: Precip of Fb, PAGE of Eb, Fb, Gb

Fb was not folded at the same time as Eb and Gb b/c we ran out of p7308. The work done to Fb is intended to follow as closely as possible that which was done to Eb and Gb.

PEG Precipitation of Fb

  • 12% PEG, 5M NaCl
  120uL rxn (3 rxns)
  240uL 20% PEG -> 12% PEG
  40uL 5M NaCl -> 0.5M NaCl
  400uL total reaction

 Ice 15 minutes
 Centrifuge @ 16G for 10 min
 Separate supernatant and pellet
   Reconstituted pellet in 16uL H2O

   Speedvaced supernatant in futile hope of reducing it to 60uL, erroneously thinking each agarose well holds 20uL (each holds 40uL)
       Reduced it to approximately 200uL, at which point it had thickened to a gel
       Reconsistuted with H2O to approximately 300uL

 Ran on 2% agarose gel, 10mM MgCl2 made by Matt two days ago
lane amount (μL) concentration component
1 7 - 1kb ladder
2 2 120uL of rxn into 16uL soln pellet
3 40 - supernatant
4 10 44nm p7308
  • gel not bright enough to really make conclusions, though bright band in lane 2 at least shows something present in pellet
  • will run again with newly made gel

    • will run PAGE w/ Eb, Fb, and Gb in streptavidin tomorrow

Titration of [MgCl2] and [oligos] in Folding Rxns

  • According to Dr. Shih's instructions, experiments varying the concentration of MgCl2 and oligos were conducted:
[MgCl2] - 10mM (final) 20mM (final) 30mM (final)
[oligos] 100nm (total) X X X
- 600nm (total) X X X

Making 10x Folding Buffers w/ Different [MgCl2]s

  • For 400uL of 200mM MgCl2 in 10x folding buffer
component [stock] uL put into soln [final]
HEPES pH 7.5 1M 200 500mM
NaCl 5M 40 500mM
MgCl2 1M 80 200mM
H2O - 80 -
  • For 400uL of 300mM MgCl2 in 10x folding buffer
component [stock] uL put into soln [final]
HEPES pH 7.5 1M 200 500mM
NaCl 5M 40 500mM
MgCl2 1M 120 300mM
H2O - 40 -

Making Different [oligos] Working Stocks

  • Because each working stock solution is made to total to 200uL, each working stock contains 12.5 rxns (=200uL/16uL).
  • To concentrate to 6x, 6 reactions worth of working stock oligos were reduced to 1 rxn worth.
    • 6 tubes (standard 1.5mL tubes) of 6rxns (96uL of working stock) were made from 3 tubes of the original working stock solution, which each total to 200uL.
      • Tubes were speedvaced for approximately 45 minutes to complete dryness; they were then reconstituted in 16uL of H2O.
  • In order to lower the amount of oligo which might be lost in moving these 16uL solutions to PCR tubes, the folding reaction components were added to the 1.5mL tubes which already contained 16uL of 6x oligos.
    • 9uL of p7308 (44nM)
    • 4uL of the appropriate 10x folding buffer (see table above).
    • 11uL of H2O
  • These were transferred to PCR tubes and folded with the FOLDINGD program.