| Making collagen solution from lyophilized rat tail collagen鼠尾制备胶原蛋白 | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-18 本站论坛 |
|  | Chen-Ming Fan Lab,Carnegie Institute of Washington
(lyophilized rat tail collagen from Roche)
DME power from Life technologies (buy the one without
NaHCO3, make a 10X solution but with the pH color indicator, and
use HCl to adjust pH to 4.0, then dilute this solution w/ water to
make the 0.1X DME solution.)
To each bottle of lyophilized collagen, add 2.5 mLs 0.1X DME pH
4.0 and 1.75 uL of a 10% acetic acid solution.
Mix well by hand until large chunks are broken up.
Mix well 1hr-O/N at 4oC on shaker.
To make collagen gels:
For every 100uL of collagen solution add 10 uL 10X DME pH 4.0
(see above note) and 0.5-1 uL of 0.8 M NaHCO3 add 0.8 M
NaHCO3 just until the solution starts to turn pink. Use this to make
a layer of collagen for your tissue. Let solidify (about 30 min at
room temp. Place you tissue on top and then lay over tissue a
second layer of collagen. Let the esecond layer solidify (30 min)
The add culture media enough to cover the top of the collagen.
This is the culture media we use:
For 5mL: (all solutions from Life Technologies)
DME/F-12 mixutre 4.5 mL
(50:50 available from Life Technologies)
Fetal Bovine Serum 0.5 mL
Glutamax 50 uL
Pen-Strep 50 uL
Non-Essential Amino Acids 50 uL
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