IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14

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(Microcon w/ detergent)
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* add 20 {{ul}} given nanostructure to center of YM-50 Micrcon tube
* add 20 {{ul}} given nanostructure to center of YM-50 Micrcon tube
* add 480 {{ul}} given folding buffer, microcentrifuge for 6 min. at 14k rcf, and repeat dilution and spinning 4 more times
* add 480 {{ul}} given folding buffer, microcentrifuge for 6 min. at 14k rcf, and repeat dilution and spinning 4 more times
 +
* yielded approx. 100 {{ul}} retentate, which was concentrated to 15 to 60 {{ul}} in a Vacufuge (about 30 min. at 45 {{c}}), depending on the sample
{| {{table}}
{| {{table}}
-
| align="center" style="background:#f0f0f0;"|'''trial'''
+
| align="center" style="background:#f0f0f0;"|'''lane'''
-
| align="center" style="background:#f0f0f0;"|'''nanostructures'''
+
| align="center" style="background:#f0f0f0;"|'''starting amt. of nanostructures'''
| align="center" style="background:#f0f0f0;"|'''wash buffer'''
| align="center" style="background:#f0f0f0;"|'''wash buffer'''
 +
| align="center" style="background:#f0f0f0;"|'''loaded onto gel'''
|-
|-
-
| 6hb-0||20 {{ul}} 6hb||1x folding buffer (10 mM {{mgcl2}})
+
| 1||||||7 {{ul}} 1 kb+ ladder
|-
|-
-
| 6hb-0.1||20 {{ul}} 6hb||1x folding buffer (10 mM {{mgcl2}}) w/ 0.1% SDS
+
| 2||||||2.25 {{ul}} p7308 and 10 {{ul}} unpurified 4.0.I
|-
|-
-
| 4-0||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}})
+
| 3||20 {{ul}} 6hb||1x folding buffer (10 mM {{mgcl2}})||half of retentate
|-
|-
-
| 4-0.01||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}}) w/ 0.01% SDS
+
| 4||20 {{ul}} 6hb||1x folding buffer (10 mM {{mgcl2}}) w/ 0.1% SDS||half of retentate
|-
|-
-
| 4-0.1||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}}) w/ 0.1% SDS
+
| 5||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}})||half of retentate
 +
|-
 +
| 6||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}}) w/ 0.01% SDS||half of retentate
 +
|-
 +
| 7||20 {{ul}} 4.0.I||1x folding buffer (10 mM {{mgcl2}}) w/ 0.1% SDS||half of retentate
|}
|}

Revision as of 15:00, 14 August 2006

Contents

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,b16 μL 250 nM9 μL4 μL 100 mM MgCl211 μL
2a,b16 μL 1.5 μM9 μL4 μL 100 mM MgCl211 μL
3a,b16 μL 250 nM9 μL4 μL 200 mM MgCl211 μL
4a,b16 μL 1.5 μM9 μL4 μL 200 mM MgCl211 μL
5a,b16 μL 250 nM9 μL4 μL 300 mM MgCl211 μL
6a,b16 μL 1.5 μM9 μL4 μL 300 mM MgCl211 μL

3. PEG & Microcon

PEG:

  • GOAL: test 8% and 10% 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 water



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
  • yielded approx. 100 μL retentate, which was concentrated to 15 to 60 μL in a Vacufuge (about 30 min. at 45 [[:Category:{{{1}}}|{{{1}}}]]), depending on the sample
lane starting amt. of nanostructures wash buffer loaded onto gel
17 μL 1 kb+ ladder
22.25 μL p7308 and 10 μL unpurified 4.0.I
320 μL 6hb1x folding buffer (10 mM MgCl2)half of retentate
420 μL 6hb1x folding buffer (10 mM MgCl2) w/ 0.1% SDShalf of retentate
520 μL 4.0.I1x folding buffer (10 mM MgCl2)half of retentate
620 μL 4.0.I1x folding buffer (10 mM MgCl2) w/ 0.01% SDShalf of retentate
720 μL 4.0.I1x folding buffer (10 mM MgCl2) w/ 0.1% SDShalf of retentate
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