There are several modifications of Dulbecco's Modified Eagle Medium (DMEM), and the actual media selected may (possibly) deviate from the original formulation. It seems important to ensure that the pH buffering capacity is properly maintained once the original pH was increased by adding NaOH. Also determinant would be the concentration of CaCl2 at the media. You may want to check my answer at the following discussion: https://www.researchgate.net/post/Can_anyone_suggest_me_the_buffer_conditions_or_methods_for_solubilising_collagen_powder_other_than_acidic_condition

I extracted my own rat tail collagen and now the gel is setting fine. Still no idea why the collagen I had bought just stopped working

Probably denatured, or wrong concentration i.e. could have been a supplier issue (or storage at your end). Interested in how you prepared your rat tail collagen (acid or pepsin), you can easily isolate rat tail collagen with acid only i.e. crosslinks are acid labile, pretty unusual for tendon and you will retain telopeptide so this will affect fibrillogenisis; which method did you use

That is odd that it works with your own extracted collagen. What method of extraction did you use?

I also have the same problem, do you solve your problem? Thanks.

Hello,

I am not sure if this answer would be still helpful or not. :) Generally in order to make collagen gel from a new stock of bottle one should always optimise the concentration of 1M NaOH. This tend to change with different stocks of collagen. You can titrate the volume of NaOH from 5ul to 14 ul and see at which concentration you get the best gel formation. Then according to that you can stick to that particular concentration for further experiments.