5 Matching Annotations
  1. May 2019
    1. Activation of H50 protein chip array
    2. b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine
    3. 5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once
    4. Typically, 100-200 ng of DNA was used in each ligation reaction. The ratio of vector to insert was maintained between 1:3 and 1:5. The reactions were usually done in a 10 μl volume containing ligation buffer (provided by the manufacturer) and 0.05 Weiss units of T4 DNA ligase, at 16°C for 12-16 hr
    5. Ligation of DNA