20210206 collagen
From mn.fysikk.laglivlab
Revision as of 13:48, 2 June 2021 by Dagkd@uio.no (talk | contribs)
- Took collagen out from freezer at 10:50
Contents
Calculating volumes for aliquoting
- concentration in vials: c= 5 mg/ml
- Thin coating = 5 ug/cm^2
- Area of coating: 2-5 cm^2 per network
- Mass of collagen: m= 5*5 = 25ug
- Volume of undiluted collagen: V = m/c = 0.025 mg/ 5 mg/ml = 0.005 ml = 5 ul, which is 1/1000 of a vial. It is completely unrealistic to handle such small volumes
- If I divide the vial volume into 25 x 200 ul it should give a reasonable amount of aliquots for thin coating
Diluting and dispensing
- Prepare sterile 17.5M acetic acid stock solution
- Dilute 1:1000 with sterile, distilled H2O
- Height of channels: 100 um = 0.01 cm
- Concentration of collagen C [ug/ml] = 5 ug/cm^2 / 100 um = 5 ug/ cm^2 / 0.01 cm = 500 ug/ml = 0.5 mg/ml => dilute 1:10 with Acetic acid
- ca 250 ul concentrated collagen + 2250 ul diluted acetic acid
- filled in a 2.5 ml syringe, added 0.6 mm diam tip and Elveflow tube.
Filling in network
- Anniken's network 100 um high was cut from PDMS
- 1.6 mm diam holes
- Plasma cleaned and assembled, tube connected
- Syringe in syringe pump, difficult to know which rate to use
- 1 ml/min - 0.1 ml/min gave too high pressure and tip flew off syringe
- 0.01 ml/min - 0.2 ml/min OK (after tube was lifted a bit off the bottom in the PDMS)
- because several disconnections (too high pressure) the tube was filled with air bubbles. These air bubbles were impossible to remove, probably due to collagen at the air-liquid interface
Suggested new procedure: fill network with pure water first and when there are no air bubbles, fill with collagen solution.