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

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 to thaw.

New folding conditions

 * on PTC-100 machine, called FOLDINGD
 * step 1: 80 for 2 min, increment -1.0 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

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 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

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 EtBr in 100 mL Tris-glycine buffer for 30 min.

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.