IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14: Difference between revisions

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* Based on [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-7|the inconclusive gels from last Monday]], the titration will be redone with c5.0 at 8% PEG, 0.5M NaCl '''final'''.
* Based on [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-7|the inconclusive gels from last Monday]], the titration will be redone with c5.0 at 8% PEG, 0.5M NaCl '''final'''.


==Mg2+, Oligo-Concentration Titration w/ c5.0==
===Goals===
* vary folding conditions ([{{mgcl2}}] and [oligo]) in order to determine best folding conditions for c5.0
* determine most efficient purification protocol (Microcon vs. PEG) based on recovery yields
===Protocol===
====1. Working Stock Concentration====
* concentrated 6 tubes of 96 {{ul}} c5.0D.L (no latches, outside-bound ligand) in Vacufuge so that [oligo]= 250nM * 6 = 1.5 {{uM}}
====2. Folding Rxns====
* used three different [[IGEM:Harvard/2006/Folding_buffers|folding buffers]] varying [{{mgcl2}}]
* used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 {{uM}} from above
* folding conditions: 80{{c}} for 2 min., decrease 1{{c}} every 2 min. for 59 more times
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Trial'''
| align="center" style="background:#f0f0f0;"|'''Oligos'''
| align="center" style="background:#f0f0f0;"|'''p7308 (44 nM)'''
| align="center" style="background:#f0f0f0;"|'''Folding Buffer (10x)'''
| align="center" style="background:#f0f0f0;"|'''Water'''
|-
| 1a,b||16 {{ul}} 250 nM||9 {{ul}}||4 {{ul}} 100 mM {{mgcl2}}||11 {{ul}}
|-
| 2a,b||16 {{ul}} 1.5 {{um}}||9 {{ul}}||4 {{ul}} 100 mM {{mgcl2}}||11 {{ul}}
|-
| 3a,b||16 {{ul}} 250 nM||9 {{ul}}||4 {{ul}} 200 mM {{mgcl2}}||11 {{ul}}
|-
| 4a,b||16 {{ul}} 1.5 {{um}}||9 {{ul}}||4 {{ul}} 200 mM {{mgcl2}}||11 {{ul}}
|-
| 5a,b||16 {{ul}} 250 nM||9 {{ul}}||4 {{ul}} 300 mM {{mgcl2}}||11 {{ul}}
|-
| 6a,b||16 {{ul}} 1.5 {{um}}||9 {{ul}}||4 {{ul}} 300 mM {{mgcl2}}||11 {{ul}}
|}
====3. PEG & Microcon====
PEG:
* GOAL: test 0% to 8% PEG precipitations
* incubated on ice for 15 min.
* spun at 16 k rcf at 4{{c}} for 10 min.
* carefully pipetted off supernatant
* resuspended "pellet" in 20 {{ul}} of respective folding buffer
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Trial'''
| align="center" style="background:#f0f0f0;"|'''Final PEG %'''
| align="center" style="background:#f0f0f0;"|'''Gel'''
| align="center" style="background:#f0f0f0;"|'''Lanes'''
| align="center" style="background:#f0f0f0;"|'''20% PEG ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''5 M NaCl ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''Nanostructures ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''Water ({{ul}})'''
| align="center" style="background:#f0f0f0;"|'''Total volume ({{ul}})'''
|-
| 1-0||0%||1||1||0||5||5||20||50
|-
| 1-2||2%||1||3||5||5||5||15||50
|-
| 1-4||4%||1||5||10||5||5||10||50
|-
| 1-6||6%||1||7||15||5||5||5||50
|-
| 1 kb+ ladder||||1||9||||||||||10
|-
| p7308||||1||10||||||||||10
|-
| 1-8||8%||1||11||20||5||5||0||50
|-
| 2-0||0%||1||13||0||5||5||20||50
|-
| 2-2||2%||1||15||5||5||5||15||50
|-
| 2-4||4%||1||17||10||5||5||10||50
|-
| 2-6||6%||1||19||15||5||5||5||50
|-
| 2-8||8%||1||21||20||5||5||0||50
|-
| 3-0||0%||1||23||0||5||5||20||50
|-
| 3-2||2%||1||25||5||5||5||15||50
|-
| 3-4||4%||1||27||10||5||5||10||50
|-
| 1 kb+ ladder||||1||29||||||||||10
|-
| p7308||||1||30||||||||||10
|-
| 3-6||6%||1||31||15||5||5||5||50
|-
| 3-8||8%||1||33||20||5||5||0||50
|-
| 4-0||0%||1||35||0||5||5||20||50
|-
| 4-2||2%||1||37||5||5||5||15||50
|-
| 4-4||4%||1||39||10||5||5||10||50
|-
| 4-6||6%||2||1||15||5||5||5||50
|-
| 4-8||8%||2||3||20||5||5||0||50
|-
| 5-0||0%||2||5||0||5||5||20||50
|-
| 5-2||2%||2||7||5||5||5||15||50
|-
| 1 kb+ ladder||||1||29||||||||||10
|-
| p7308||||1||30||||||||||10
|-
| 5-4||4%||2||11||10||5||5||10||50
|-
| 5-6||6%||2||13||15||5||5||5||50
|-
| 5-8||8%||2||15||20||5||5||0||50
|-
| 6-0||0%||2||17||0||5||5||20||50
|-
| 6-2||2%||2||19||5||5||5||15||50
|-
| 6-4||4%||2||21||10||5||5||10||50
|-
| 6-6||6%||2||23||15||5||5||5||50
|-
| 6-8||8%||2||25||20||5||5||0||50
|-
| 1 kb+ ladder||||1||29||||||||||10
|-
| p7308||||1||30||||||||||10
|}


==Microcon w/ detergent==
==Microcon w/ detergent==

Revision as of 10:50, 14 August 2006

Goals for today

Microcon Purification Tweaking

  • repeat Friday's mega PEG ppt on 5.0 (?)
  • Micron experiments with 0.1% and 0.01% SDS in buffer
    • ...and use 1x folding buffer and not water for washes
    • also: perform control expt with 10 bp+ ladder, since according to Millipore documentation, the filter should retain ds DNAs longer than 100 bp

Streptavidin-Bead "Protection" Assay on Inside- and Outside-Biotinylated c5.0

  • NB: no good purification of nanostructure from oligo has been achieved, but gel separation after elution should differentiate formerly bead-bound oligos from formerly bead-bound nanostructures

Redux of [Mg++], [oligos]



Mg2+, Oligo-Concentration Titration w/ c5.0

Goals

  • vary folding conditions ([MgCl2] and [oligo]) in order to determine best folding conditions for c5.0
  • determine most efficient purification protocol (Microcon vs. PEG) based on recovery yields

Protocol

1. Working Stock Concentration

  • concentrated 6 tubes of 96 μL c5.0D.L (no latches, outside-bound ligand) in Vacufuge so that [oligo]= 250nM * 6 = 1.5 μM

2. Folding Rxns

  • used three different folding buffers varying [MgCl2]
  • used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 μM from above
  • folding conditions: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min., decrease 1[[:Category:{{{1}}}|{{{1}}}]] every 2 min. for 59 more times
Trial Oligos p7308 (44 nM) Folding Buffer (10x) Water
1a,b 16 μL 250 nM 9 μL 4 μL 100 mM MgCl2 11 μL
2a,b 16 μL 1.5 μM 9 μL 4 μL 100 mM MgCl2 11 μL
3a,b 16 μL 250 nM 9 μL 4 μL 200 mM MgCl2 11 μL
4a,b 16 μL 1.5 μM 9 μL 4 μL 200 mM MgCl2 11 μL
5a,b 16 μL 250 nM 9 μL 4 μL 300 mM MgCl2 11 μL
6a,b 16 μL 1.5 μM 9 μL 4 μL 300 mM MgCl2 11 μL

3. PEG & Microcon

PEG:

  • GOAL: test 0% to 8% PEG precipitations
  • incubated on ice for 15 min.
  • spun at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipetted off supernatant
  • resuspended "pellet" in 20 μL of respective folding buffer
Trial Final PEG % Gel Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) Water (μL) Total volume (μL)
1-0 0% 1 1 0 5 5 20 50
1-2 2% 1 3 5 5 5 15 50
1-4 4% 1 5 10 5 5 10 50
1-6 6% 1 7 15 5 5 5 50
1 kb+ ladder 1 9 10
p7308 1 10 10
1-8 8% 1 11 20 5 5 0 50
2-0 0% 1 13 0 5 5 20 50
2-2 2% 1 15 5 5 5 15 50
2-4 4% 1 17 10 5 5 10 50
2-6 6% 1 19 15 5 5 5 50
2-8 8% 1 21 20 5 5 0 50
3-0 0% 1 23 0 5 5 20 50
3-2 2% 1 25 5 5 5 15 50
3-4 4% 1 27 10 5 5 10 50
1 kb+ ladder 1 29 10
p7308 1 30 10
3-6 6% 1 31 15 5 5 5 50
3-8 8% 1 33 20 5 5 0 50
4-0 0% 1 35 0 5 5 20 50
4-2 2% 1 37 5 5 5 15 50
4-4 4% 1 39 10 5 5 10 50
4-6 6% 2 1 15 5 5 5 50
4-8 8% 2 3 20 5 5 0 50
5-0 0% 2 5 0 5 5 20 50
5-2 2% 2 7 5 5 5 15 50
1 kb+ ladder 1 29 10
p7308 1 30 10
5-4 4% 2 11 10 5 5 10 50
5-6 6% 2 13 15 5 5 5 50
5-8 8% 2 15 20 5 5 0 50
6-0 0% 2 17 0 5 5 20 50
6-2 2% 2 19 5 5 5 15 50
6-4 4% 2 21 10 5 5 10 50
6-6 6% 2 23 15 5 5 5 50
6-8 8% 2 25 20 5 5 0 50
1 kb+ ladder 1 29 10
p7308 1 30 10

Microcon w/ detergent

  • add 20 μL given nanostructure to center of YM-50 Micrcon tube
  • add 480 μL given folding buffer, microcentrifuge for 6 min. at 14k rcf, and repeat dilution and spinning 4 more times
trial nanostructures wash buffer
6hb-0 20 μL 6hb 1x folding buffer (10 mM MgCl2)
6hb-0.1 20 μL 6hb 1x folding buffer (10 mM MgCl2) w/ 0.1% SDS
4-0 20 μL 4.0.I 1x folding buffer (10 mM MgCl2)
4-0.01 20 μL 4.0.I 1x folding buffer (10 mM MgCl2) w/ 0.01% SDS
4-0.1 20 μL 4.0.I 1x folding buffer (10 mM MgCl2) w/ 0.1% SDS
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