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Team:HKU-HKBU/Motor Preliminary Trials
From 2009.igem.org
Contents |
Membrane Production
Step 1
The Immobolin-P membrane was first made wet and consequently homogenized with a mini-homogenizer.
Results: The membrane could not be broken into small pieces.
Step 2
The Immobolin-P membrane was first moistened, then it was put into a 10ml centrifugation tube. The tube was then totally filled with glass beads, and undergo vortexing subsequently.
Results: The membrane remained intact.
Step 3
The Immobolin-P membrane was moistened and liquefied nitrogen was poured onto it. The membrane was broken into pieces by human hands.
Results: The membrane remained intact.
Step 4
The Immobolin-P membrane was first biotinylated and cut into very small pieces by human hands. Then, the membrane fragments were put into a mould made with aluminium foil and fixed into it with the help of glue. The mould along with the membrane fragment were cut with a Leica-crytomicrotome into further smaller pieces the size of 100umx60umx100um.
Step 5
Some elemental silicon fragments were put inside a 1-ml eppendorf tube. The protein-biotin complex and some concentrated HCL were then added into the same tube. The tube was put inside a water bath for 2 hours. The silicon fragments were made dry by rinsing with PBS and followed by air drying. Then, some streptavidin containing beads were added onto the fragments. The fragments were observed under a microscope.
Results: No beads were bound to the silicon fragments.
Step 6
The silicon fragments were silanized by soaking them for 2 h in a solution of aminopropyl triethoxyl silane (3% aminopropyl triethoxyl silane, 2% acetic acid, 5% water, 90% ethanol), then rinsed with ethanol, and dried with a PCR machine for 5 mins. The amino-coated rotors (Fig. 2Bh) were then reacted with 1 mM succinimidyl-6-(biotinamido)-6-hexana- mido hexanoate (EZ-Link NHS-LC-LC-biotin; Pierce, Rock- ford, IL) dissolved in 40 mM phosphate buffer (pH 8.0) for 1 h at 37°C. Then strepatavidin beads were allowed to bind onto it and the fragments were observed under a microscope.
