IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1: Difference between revisions

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** determine lower threshold of imaging
** determine lower threshold of imaging


==SYBR gold testing==
* goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
* methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with [http://probes.invitrogen.com/media/pis/mp11494.pdf Invitrogen SYBR GOld].
** prepared 1x solution: 10 {{ul}} thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4{{c}}.
{| {{table}}
| align="center" rowspan=2 style="background:#f0f0f0;"|'''lane'''
| align="center" colspan=3 style="background:#f0f0f0;"|'''DNA''' (3.2.7.2b)<br>(12.5 pg DNA / fmol)
|-
| align="center"|({{ul}})|| align="center"|(fmols)|| align="center"|(pg)
|-
| 1||15 {{ul}} 1 {{um}}||15,000||187,500
|-
| 2||10 {{ul}} 1 {{um}}||10,000||125,000
|-
| 3||5 {{ul}} 1 {{um}}||5,000||62,500
|-
| 4||2.5 {{ul}} 1.0 {{um}}||2,500||31,250
|-
| 5||1 {{ul}} 1.0 {{um}}||1,000||12,500
|-
| 6||5 {{ul}} 0.1 {{um}}||500||6,250
|-
| 7||2.5 {{ul}} 0.1 {{um}}||250||3,125
|-
| 8||1 {{ul}} 0.1 {{um}}||100||1,250
|-
| 9||5 {{ul}} 0.01 {{um}}||50||625
|-
| 10||2.5 {{ul}} 0.01 {{um}}||25||313
|-
| 11||1 {{ul}} 0.01 {{um}}||10||125
|-
| 12|| colspan="3"|2 {{ul}} 1kb+ ladder
|}


==PEG precipitation==
==PEG precipitation==

Revision as of 09:10, 1 August 2006

To do today

  • ordering
    • new oligo ligands
    • AscIII (if not already ordered
  • PEG precipitations
    • repeat experiment, run on PA gel, run on agarose gel
  • SYBR gold
    • read technical specs
    • determine lower threshold of imaging

SYBR gold testing

  • goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
  • methods: 12% PA gel with lanes listed below, run for 20 min. at 130 V, then stained with Invitrogen SYBR GOld.
    • prepared 1x solution: 10 μL thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4[[:Category:{{{1}}}|{{{1}}}]].
lane DNA (3.2.7.2b)
(12.5 pg DNA / fmol)
(μL) (fmols) (pg)
1 15 μL 1 μM 15,000 187,500
2 10 μL 1 μM 10,000 125,000
3 5 μL 1 μM 5,000 62,500
4 2.5 μL 1.0 μM 2,500 31,250
5 1 μL 1.0 μM 1,000 12,500
6 5 μL 0.1 μM 500 6,250
7 2.5 μL 0.1 μM 250 3,125
8 1 μL 0.1 μM 100 1,250
9 5 μL 0.01 μM 50 625
10 2.5 μL 0.01 μM 25 313
11 1 μL 0.01 μM 10 125
12 2 μL 1kb+ ladder

PEG precipitation

  • Goal: repeat titration of PEG precipitation conditions for folded containers
  • Folding reactions
    • 2 samples of 6hb (100 μL final volume)
    • 4 samples of design 5 barrels (100 μL volume)
    • Mix the following
      • 10 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2
      • 40 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
      • 50 uL p7308 20 nM (10 nM final concentration)
    • Anneal from 80[[:Category:{{{1}}}|{{{1}}}]] to 20[[:Category:{{{1}}}|{{{1}}}]], -1[[:Category:{{{1}}}|{{{1}}}]] per min
  • PEG precipitations
  • Plan to use 200 μL final volume
  • Trying 4%, 6%, 8%, 10%, 12%, 14%
  • Add 40 μL 10 nM scaffold/100 nM oligo folded mix
  • Add 20% PEG solution (40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL
  • Add 5 M NaCl stock solution (20 μL)
  • Gel Analysis
    • Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
    • Load 1kb ladder (1 lane)
    • Load c5 supernatant, pellet for 0%, 4%, 6%, 8%, 10%, 12%, 14% (13 lanes)
    • Load 6hb supernatant, pellet for 0%, 4%, 14% (5 lanes)
    • 19 lanes total
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