Making collagen solution from lyophilized rat tail collagen
Chen-Ming Fan Lab,Carnegie Instituteof Washington
(lyophilized rat tail collagen fromRoche)
DME power from Life technologies (buy the one without NaHCO3, makea 10X solution but with the pH color indicator, and
use HCl to adjust pH to 4.0, then dilute this solution w/ water tomake the 0.1X DME solution.)
To each bottle of lyophilized collagen, add 2.5 mLs 0.1X DME pH4.0and 1.75 uL of a 10% acetic acid solution.Mix well by hand untillarge 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(seeabove 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 tomake a layer of collagen for your tissue. Let solidify (about 30min at room temp. Place you tissue on top and then lay over tissuea 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 4.5mL(50:50 available from Life Technologies)
Fetal Bovine Serum 0.5 mL Glutamax 50 uL Pen-Strep 50uLNon-Essential Amino Acids 50 uL