Biomedical Engineering Reference
In-Depth Information
nucleic acids onto QIAEX II silica gel particles in the presence
of chaotropic salt. It is designed for batch purifi cation of a wide
range of DNA fragment sizes from 40 bps to 50 kb.
21. If the sequencing trace gives an indication that two PCR frag-
ments are mixed, these fragments can be separated by cloning
in a T/A cloning vector and sequencing several colonies to
identify the fragments.
22. It is important that the medium and agar are not warmer than
41 °C, as the cells might be subjected to heat shock.
23. It is important to keep the fi nal concentration of the top agar
close to 0.36 %. If the percentage of agar is low, the top agar
layer will be very soft, and the spheroids could be removed
when the medium is being refreshed. So the volume of
10A-medium can be adjusted accordingly.
24. Do one tube at a time; otherwise, the agar may solidify before
it is overlaid in the 6-well dish. Do not keep the cell-medium-
agar mixture at 41 °C. The cells will lose their viability and will
give a lower spheroid count.
25. It may be diffi cult to remove the medium from the top agar
completely. In this case, remove half of the old medium and
replace it with the same volume of fresh medium.
26. MTT is a colorimetric biochemical reaction used for assessing
the viability of the cells. It utilizes the electrons produced by
the mitochondria to reduce the yellow tetrazolium dye
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
to its insoluble formazan compound, which has a purple color,
thus staining the cells or spheroids purple.
27. The pH of 10mM KH
2
PO
4
is 4.68 which changes the phenol
red dye in the medium from red to yellow. This will contrast
better with the colonies for scanning or photography
documentation.
28. For a lysate volume of 200
L of 6× dye is added to
make a 1× dye mixture. An alternative, rough cell lysis method
is to use the dye, diluted to 2× concentration and added to the
cells directly. This is a rough protein lysate preparation and
does not allow for normalization of the protein expression
against the starting cell line. The Laemmli buffer will lyse the
cells entirely, and the DNA will be released. Hence, the lysate
at this stage will be very viscous. The viscosity will disappear
after the 95 °C heat treatment for 10 min. Some researchers
like to heat the samples twice for a period of 5 min each with
brief vortexing of the sample in between the two treatments.
29. The presence of air in acrylamide prevents the cross-linking
process. In some protocols, degassing of the acrylamide mix-
ture is performed before APS and TEMED are added.
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