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

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.