IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-13: Difference between revisions

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==Creating pre-working stocks and working stocks from design 3.2 oligos==
==Container 3.2==


'''Mix 10 {{ul}} of each 50 {{um}} stock oligo'''
====Initial Container v3.2 Preparation and Analysis====
* c3.2.1 core barrel: 3.2p1A01–3.2p1H10 (94 total)
*Oligos arrived this morning from Invitrogen.  Plan for today is to mix oligos together into pre-stocks, and then working stocks, and then try some initial folding experiments and gel analysis.
* c3.2.2 core top lid: 3.2p2A01–3.2p2C04 (28 total)
*Oligos were ''very'' frozen and were left overnight at 4{{c}} to thaw.
* c3.2.3 core bottom lid: 3.2p2C05–3.2p2E10 (30 total)
 
* c3.2.4 barrel at inside apatamer locations -aptamers: 3.2p2E11–3.2p2F01 (3 total)
====New folding conditions====
* c3.2.5 barrel at outside aptamer locations -aptamers: 3.2p2F02–3.2p2F04 (3 total)
 
* c3.2.6 barrel at inside apatamer locations +aptamers: 3.2p2F05–3.2p2F07 (3 total)
* on PTC-100 machine, called FOLDINGD
* c3.2.7 barrel at outside aptamer locations +aptamers: 3.2p2F08–3.2p2F10 (3 total)
* step 1: 80{{c}} for 2 min, increment -1.0{{c}} each cycle
* c3.2.8 barrel at latch locations -latches: 3.2p2F11–3.2p2F12 (2 total)
* step 2: go to step 1, 59 more times
* c3.2.9 lid at latch locations -latches (empty):
* step 3: end
* c3.2.10 barrel at latch locations +latch1 +latch2: 3.2p2G01–3.2p2G02 (2 total)
 
* c3.2.11 latch from barrel +latch1 -latch2: 3.2p2G03–3.2p2G04 (2 total)
====Ordered 3'-biotinylated oligos====
* c3.2.12 latch from barrel -latch1 +latch2: 3.2p2G05–3.2p2G06 (2 total)
 
* c3.2.13 lid at latch locations +latch1 +latch2 (empty):
*...because we expect biotin-streptavidin binding to be more reliable than thrombin-aptamer binding
* c3.2.14 latch from lids +latch1 -latch2: 3.2p2G07–3.2p2G08 (2 total)
 
* c3.2.15 latch from lids -latch1 +latch2: 3.2p2G09–3.2p2G10 (2 total)
c.3.2.6.1b      GGAATAGGGAACCTATTATTCACCCTCAGAGCCACTTTCATCTTT-biotin
* c3.2.16 latch staples +latch2: 3.2p2G11–3.2p2H02 (4 total)
c.3.2.6.2b      AAGCACTAGTAAAAGAGTCTGACTTGCCTGAGTAGACAGAGGTTT-biotin
* c3.2.17 displacement strands latch1: 3.2p2H03–3.2p2H06 (4 total)
c.3.2.6.3b      AGTCAGAAGCAAAGCGGATTGGTAATAGTAAAATGTTT-biotin
* c3.2.18 displacement strands latch2: 3.2p2H07–3.2p2H10 (4 total)
c.3.2.7.1b      GGCAAAAATCAGCTTGCTTTCTTTCAACAGTTTCAATAGCCCTTT-biotin
c.3.2.7.2b      TCAGATGATGGCAATTCATCACACCTTGCTGAACCTTT-biotin
c.3.2.7.3b      TTTATCCTCTTTCCAGAGCCTTTT-biotin
 
==Protein & DNA-staining Experiment (Repeat 1)==
1. Rxn Mixtures
 
    {| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube'''
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Thrombin mass''' ({{ug}})
| align="center" style="background:#f0f0f0;"|'''2 {{um}} thrombin '''({{ul}})<br>(2 {{uM}} = 1.299 {{ug}}/{{ul}})
| align="center" style="background:#f0f0f0;"|'''water''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''10x loading dye''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''total volume''' ({{ul}})
|-
| 1||8||0.65||0.5||6.5||3.0||10.0
|-
| 2||9||1.30||1.0||6.0||3.0||10.0
|-
| 3||10||1.95||1.5||5.5||3.0||10.0
|-
| 4||11||2.69||2.0||5.0||3.0||10.0
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube'''
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''2 {{um}} Aptamer''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''water''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''10x loading dye''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''total volume''' ({{ul}})
|-
| 1||2||1.0||6.0||3.0||10.0
|-
| 2||3||2.0||5.0||3.0||10.0
|-
| 3||4||3.0||4.0||3.0||10.0
|-
| 4||5||4.0||3.0||3.0||10.0
|}
 
2. Protocol
    - 12% Invitrogen polyacrylamide gel run @ 120V for 1.25 hrs.  
    - gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom)
    - protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 1 hr.
    - DNA section stained w/ 10{{ul}} EtBr in 100 mL Tris-glycine buffer for 1 hr.
 
3. Results
    - loading dye moved about 2/3 of the way down the gel
    - protein did not image
    - small spot of DNA in lane 4
 
==Protein & DNA-staining Experiment (Repeat 2)==
1. Rxn Mixtures
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube'''
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''Thrombin mass''' ({{ug}})
| align="center" style="background:#f0f0f0;"|'''2 {{um}} thrombin '''({{ul}})<br>(2 {{uM}} = 1.299 {{ug}}/{{ul}})
| align="center" style="background:#f0f0f0;"|'''water''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''10x loading dye''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''total volume''' ({{ul}})
|-
| 0||1||0||0.0||7.0||3.0||10.0
|-
| 1||2||0.65||0.5||6.5||3.0||10.0
|-
| 2||3||1.30||1.0||6.0||3.0||10.0
|-
| 3||4||1.95||1.5||5.5||3.0||10.0
|-
| 4||5||2.69||2.0||5.0||3.0||10.0
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube'''
| align="center" style="background:#f0f0f0;"|'''Lane'''
| align="center" style="background:#f0f0f0;"|'''2 {{um}} Aptamer''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''water''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''10x loading dye''' ({{ul}})
| align="center" style="background:#f0f0f0;"|'''total volume''' ({{ul}})
|-
| 0||7||0.0||7.0||3.0||10.0
|-
| 1||8||1.0||6.0||3.0||10.0
|-
| 2||9||2.0||5.0||3.0||10.0
|-
| 3||10||3.0||4.0||3.0||10.0
|-
| 4||11||4.0||3.0||3.0||10.0
|}
 
2. Protocol
*12% Invitrogen polyacrylamide gel run @ 120V for 15 min.
*gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom)
*protein section stained w/ GelCode Blue Stain (Coomassie Blue) (the same Coomassie Blue used in repeat 1) for 30 min.
*DNA section stained w/ 10{{ul}} EtBr in 100 mL Tris-glycine buffer for 30 min.
 
[[Image:20060712_thrombin.jpg|thumb|Thrombin, 12% PAGE]][[Image:20060712_aptamers.jpg|thumb|ss DNA, 12% PAGE]]3. Results
*both protein and DNA successfully imaged
*protein appears to be in distinct bands, implying that there are several different sized molecules in the protein mixture
*perhaps the fastest band is thrombin monomer, the second fastest is dimer, etc.

Latest revision as of 19:24, 18 July 2006

Container 3.2

Initial Container v3.2 Preparation and Analysis

  • Oligos arrived this morning from Invitrogen. Plan for today is to mix oligos together into pre-stocks, and then working stocks, and then try some initial folding experiments and gel analysis.
  • Oligos were very frozen and were left overnight at 4[[:Category:{{{1}}}|{{{1}}}]] to thaw.

New folding conditions

  • on PTC-100 machine, called FOLDINGD
  • step 1: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min, increment -1.0[[:Category:{{{1}}}|{{{1}}}]] each cycle
  • step 2: go to step 1, 59 more times
  • step 3: end

Ordered 3'-biotinylated oligos

  • ...because we expect biotin-streptavidin binding to be more reliable than thrombin-aptamer binding
c.3.2.6.1b      GGAATAGGGAACCTATTATTCACCCTCAGAGCCACTTTCATCTTT-biotin
c.3.2.6.2b      AAGCACTAGTAAAAGAGTCTGACTTGCCTGAGTAGACAGAGGTTT-biotin
c.3.2.6.3b      AGTCAGAAGCAAAGCGGATTGGTAATAGTAAAATGTTT-biotin
c.3.2.7.1b      GGCAAAAATCAGCTTGCTTTCTTTCAACAGTTTCAATAGCCCTTT-biotin
c.3.2.7.2b      TCAGATGATGGCAATTCATCACACCTTGCTGAACCTTT-biotin
c.3.2.7.3b      TTTATCCTCTTTCCAGAGCCTTTT-biotin

Protein & DNA-staining Experiment (Repeat 1)

1. Rxn Mixtures

Tube Lane Thrombin mass (μg) 2 μM thrombin (μL)
(2 μM = 1.299 μg/μL) 		water (μL) 10x loading dye (μL) total volume (μL)
1 8 0.65 0.5 6.5 3.0 10.0
2 9 1.30 1.0 6.0 3.0 10.0
3 10 1.95 1.5 5.5 3.0 10.0
4 11 2.69 2.0 5.0 3.0 10.0
Tube Lane 2 μM Aptamer (μL) water (μL) 10x loading dye (μL) total volume (μL)
1 2 1.0 6.0 3.0 10.0
2 3 2.0 5.0 3.0 10.0
3 4 3.0 4.0 3.0 10.0
4 5 4.0 3.0 3.0 10.0

2. Protocol 

   - 12% Invitrogen polyacrylamide gel run @ 120V for 1.25 hrs. 
   - gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom)
   - protein section stained w/ GelCode Blue Stain (Coomassie Blue) for 1 hr.
   - DNA section stained w/ 10μL EtBr in 100 mL Tris-glycine buffer for 1 hr.

3. Results 

   - loading dye moved about 2/3 of the way down the gel
   - protein did not image
   - small spot of DNA in lane 4

Protein & DNA-staining Experiment (Repeat 2)

1. Rxn Mixtures

Tube Lane Thrombin mass (μg) 2 μM thrombin (μL)
(2 μM = 1.299 μg/μL) 		water (μL) 10x loading dye (μL) total volume (μL)
0 1 0 0.0 7.0 3.0 10.0
1 2 0.65 0.5 6.5 3.0 10.0
2 3 1.30 1.0 6.0 3.0 10.0
3 4 1.95 1.5 5.5 3.0 10.0
4 5 2.69 2.0 5.0 3.0 10.0
Tube Lane 2 μM Aptamer (μL) water (μL) 10x loading dye (μL) total volume (μL)
0 7 0.0 7.0 3.0 10.0
1 8 1.0 6.0 3.0 10.0
2 9 2.0 5.0 3.0 10.0
3 10 3.0 4.0 3.0 10.0
4 11 4.0 3.0 3.0 10.0

2. Protocol

  • 12% Invitrogen polyacrylamide gel run @ 120V for 15 min.
  • gel cut into DNA & protein sections after removal from cartridge (slight tearing towards bottom)
  • protein section stained w/ GelCode Blue Stain (Coomassie Blue) (the same Coomassie Blue used in repeat 1) for 30 min.
  • DNA section stained w/ 10μL EtBr in 100 mL Tris-glycine buffer for 30 min.
Thrombin, 12% PAGE
ss DNA, 12% PAGE

3. Results

  • both protein and DNA successfully imaged
  • protein appears to be in distinct bands, implying that there are several different sized molecules in the protein mixture
  • perhaps the fastest band is thrombin monomer, the second fastest is dimer, etc.
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