IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-13

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Contents

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)
180.650.56.53.010.0
291.301.06.03.010.0
3101.951.55.53.010.0
4112.692.05.03.010.0
Tube Lane 2 μM Aptamer (μL) water (μL) 10x loading dye (μL) total volume (μL)
121.06.03.010.0
232.05.03.010.0
343.04.03.010.0
454.03.03.010.0

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)
0100.07.03.010.0
120.650.56.53.010.0
231.301.06.03.010.0
341.951.55.53.010.0
452.692.05.03.010.0
Tube Lane 2 μM Aptamer (μL) water (μL) 10x loading dye (μL) total volume (μL)
070.07.03.010.0
181.06.03.010.0
292.05.03.010.0
3103.04.03.010.0
4114.03.03.010.0

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
Thrombin, 12% PAGE
ss DNA, 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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