IGEM:Harvard/2006/Adaptamers/Notebook/2006-7-25

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7/25

Low Mass Ladder [invitrogen]: 6 blunt-ended fragments from 100bp to 2000 bp Lambda DNA HindIII (lambda digested by HindIII) [neb]: 23130, 94116, ..., 125 bp

Morning: the big human thrombin experiment.

8% gel run at 120V for 1.5 hours.

lane 1: ladder

lane 2: human thrombin

lane 3: human thrombin + T5

lane 4: human thrombin + denatured T5

lane 5: bovine thrombin

lane 6: bovine thrombin + T5

lane 7: bovine thrombin + denatured T5

lane 8: ladder

lane 9: T5

lane 10: denatured T5

lane 11: denatured T5+ bovine

lane 12: denatured T5+ human

Image:cst725morningprotein.jpg Image:cst725morningdna.jpg

I also attempted to see if the DNA components of our aptamers were capable of binding each other. Each lane contains 40 pmol of oligo or complex. Ran a 12% gel for 1 hour at 120V. Incubation was for 15 minutes total. Denaturation: oligos mixed, heated to 90 C for 5 minutes, then rapidly cooled to 4 C for 5 minutes, followed by 5 minutes at room temperature.

Lane 1: ladder

Lane 2: T20

Lane 3: S20

Lane 4: T20 + S20

Lane 5: T20 + S20; denatured during incubation

Lane 6: T35

Lane 7: S35

Lane 8: T35+S35

Lane 9: T35 + S35; denatured during incubation

Lane 10: T50

Lane 11: S50

Lane 12: T50 + S50 (denaturation condition)

Image:cst725afternoondna.jpg

The complementary sections appeared to have come together at a pretty high rate, both for denaturing and non-denaturing conditions.

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