IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-2: Difference between revisions

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==SYBR Gold and silver staining==
==SYBR Gold and silver staining==


* goal: see how little ssDNA we can image with both SYBR gold and silver staining
{| {{table}}
{| {{table}}
| align="center" rowspan=2 style="background:#f0f0f0;"|'''lane'''
| align="center" rowspan=2 style="background:#f0f0f0;"|'''lane'''
Line 69: Line 70:
| align="center"|({{ul}})|| align="center"|(fmols)|| align="center"|(pg)
| align="center"|({{ul}})|| align="center"|(fmols)|| align="center"|(pg)
|-
|-
| 1||15 {{ul}} 1 {{um}}||15,000||209,000
| 1||15 {{ul}} 1 {{um}}||15,000||209,000||0
|-
|-
| 2||10 {{ul}} 1 {{um}}||10,000||139,000
| 2||10 {{ul}} 1 {{um}}||10,000||139,000||5
|-
|-
| 3||5 {{ul}} 1 {{um}}||5,000||69,500
| 3||5 {{ul}} 1 {{um}}||5,000||69,500||10
|-
|-
| 4||2.5 {{ul}} 1.0 {{um}}||2,500||34,800
| 4||2.5 {{ul}} 1.0 {{um}}||2,500||34,800||12.5
|-
|-
| 5||1 {{ul}} 1.0 {{um}}||1,000||13,900
| 5||1 {{ul}} 1.0 {{um}}||1,000||13,900||14
|-
|-
| 6||5 {{ul}} 0.1 {{um}}||500||6,950
| 6||5 {{ul}} 0.1 {{um}}||500||6,950||10
|-
|-
| 7||2.5 {{ul}} 0.1 {{um}}||250||3,480
| 7||2.5 {{ul}} 0.1 {{um}}||250||3,480||12.5
|-
|-
| 8||1 {{ul}} 0.1 {{um}}||100||1,390
| 8||1 {{ul}} 0.1 {{um}}||100||1,390||14
|-
|-
| 9||5 {{ul}} 0.01 {{um}}||50||695
| 9||5 {{ul}} 0.01 {{um}}||50||695||10
|-
|-
| 10||2.5 {{ul}} 0.01 {{um}}||25||348
| 10||2.5 {{ul}} 0.01 {{um}}||25||348||12.5
|-
|-
| 11||1 {{ul}} 0.01 {{um}}||10||139
| 11||1 {{ul}} 0.01 {{um}}||10||139||14
|-
|-
| 12|| colspan="3"|2 {{ul}} 1kb+ ladder
| 12|| colspan="3"|10 {{ul}} 1kb+ ladder||5
|}
|}


Revision as of 10:36, 2 August 2006

PEG precipitation

  • Goal: repeat titration of PEG precipitation conditions for 6hb
  • Folding reactions
    • 6 samples of 6hb (100 μL final 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 3%, 4%, 5%, 10%, 14%
  • Add 40 μL 10 nM scaffold/100 nM oligo folded mix
  • Add 20% PEG solution (30 μL, 40 μL, 50 μL, 100 μL, 140 μL)
  • Add 5 M NaCl stock solution (20 μL)
  • Add as much water to each as it takes to get them to a 200 μL final volume
  • Incubate on ice for 15 minutes
  • Spin 16k rcf for 10 minutes
  • Pipette out supernatant into separate tube
  • Resuspend pellet in 1x folding buffer volume equal to the supernatant
  • Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM MgCl2)
  • 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
File:IGEM06-080206.jpg
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Dye
0 1kb DNA ladder (4 μL) 0 μL
1 6hb untreated (10 μL) 2 μL
2 6hb 3% PEG supernatant (10 μL) 2 μL
3 6hb 3% PEG pellet (10 μL) 2 μL
4 6hb 4% PEG supernatant (10 μL) 2 μL
5 6hb 4% PEG pellet (10 μL) 2 μL
6 6 hb 5% PEG supernatant (10 μL) 2 μL
7 6 hb 5% PEG pellet (10 μL) 2 μL
8 6 hb 10% PEG supernatant (10 μL) 2 μL
9 6 hb 10% PEG pellet (10 μL) 2 μL
10 6 hb 14% PEG supernatant (10 μL) 2 μL
11 6 hb 14% PEG pellet (10 μL) 2 μL

SYBR Gold and silver staining

  • goal: see how little ssDNA we can image with both SYBR gold and silver staining
lane DNA (3.2.8.1)
(13.9 pg DNA / fmol) 		water (μL)
(μL) (fmols) (pg)
1 15 μL 1 μM 15,000 209,000 0
2 10 μL 1 μM 10,000 139,000 5
3 5 μL 1 μM 5,000 69,500 10
4 2.5 μL 1.0 μM 2,500 34,800 12.5
5 1 μL 1.0 μM 1,000 13,900 14
6 5 μL 0.1 μM 500 6,950 10
7 2.5 μL 0.1 μM 250 3,480 12.5
8 1 μL 0.1 μM 100 1,390 14
9 5 μL 0.01 μM 50 695 10
10 2.5 μL 0.01 μM 25 348 12.5
11 1 μL 0.01 μM 10 139 14
12 10 μL 1kb+ ladder 5
  • loaded in two gels
  • ran at 130V for 30 min.
  • stained on in silver stain, the other in SYBR gold
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