Difference between revisions of "20210319 3Dcell labnotes"
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=== Filling with water and gel === | === Filling with water and gel === | ||
| − | In room 431 | + | In laf bench in room 431, or maybe in 118 is OK? |
| − | * fill | + | It should be fairly sterile as well... If we wait long time between bonding the circuit and filling it the PDMS will become hydrophobic. The main idea of the procedure is that the PDMS should be slightly hydrophobic, but not too much to get the gel into the side chambers, but not to pass the column. |
| + | |||
| + | Idea for gel filling on/under microscope. | ||
| + | * <span style="color:#ff0000"> collagen preparations need ice!!!! Ice is available in the chemistry department</span> | ||
| + | * plug non-hydrogel inlets/outlets | ||
| + | * connect output to syringe with air to allow control of high or low pressure | ||
| + | * fill ungelled solution in pipette | ||
| + | * enter pipette in input tube and press solution through to outlet | ||
| + | * if solution does not enter lamella, increase pressure at outlet and keep pressing in solution, increase pressure until lamella are filled | ||
| + | * remove pipette and gently aspire gel solution from channel, leaving gel in lamellae | ||
| + | |||
| + | Questions: | ||
| + | * How to control inflow of such small amounts (1-10 ul)? | ||
| + | * A pipette is perhaps too brutal? | ||
| + | * Test with water in another circuit first? | ||
| + | * should I use the pressure control system? | ||
| + | ** aspire 10 ul into tube with pipette | ||
| + | ** couple tube to pressure control | ||
| + | ** increase pressure until it flows through main channel | ||
| + | ** increase pressure on opposite side to stop main flow and press into lamellae | ||
| + | ** when lamella are filled | ||
| + | *** either press harder with back pressure/ release pressure of pressure control | ||
| + | *** or aspire on back end | ||
| + | ** to fill main channel with air | ||
| + | * small volume syringe | ||
| + | ** is probably precise enough? | ||
| + | ** fill 1-2 cm of tube with solution. How do I do that? If this is 10 ul I can control this with a pipette or use our special syringes! | ||
| + | ** syringe to aspire, to fill, then counter-pressure to fill lamella, then draw solution back into syringe | ||
| + | * fill main channel with DMEM media or PBS | ||
=== Aliquoting === | === Aliquoting === | ||
| − | * Should be in final volume samples | + | * Should be in final volume samples. How large? For gels in mufluid I now need max 2mm x 10mm x 0.1 mm = 2 mm^3 = 2 ul plus the entry volumes. Entry volumes: Ø 1 mm tube x 10 mm = 10 ul. This means that 30 ul is much more than enough! |
| + | * As a first trial, [https://ibidi.com/img/cms/support/AN/AN26_CollagenI_protocols.pdf Ibid recommends] using a 1.5 mg/ml Collagen I gel | ||
* Pure or diluted | * Pure or diluted | ||
** Pure can be diluted to whatever later | ** Pure can be diluted to whatever later | ||
| + | ** I only need 9 ul to make 30 ul ready collagen. How do I store such small amounts? Is that practical? | ||
| + | ** Larger volumes will only end in waste... | ||
** Diluted | ** Diluted | ||
*** is a larger volume and easier to handle | *** is a larger volume and easier to handle | ||
*** Dilute 99-100% acetic acid to 17.5 mM. How? | *** Dilute 99-100% acetic acid to 17.5 mM. How? | ||
*** Need DI water (new) for dilution | *** Need DI water (new) for dilution | ||
Latest revision as of 18:43, 19 March 2021
Contents
Casting PDMS 10:1
- poured about 40 ml into disposable beaker on the balance: 32.2g
- poured curing agent by hand until weight was 35.4 g
- mixed. lots of bubbles in mixture
- put beaker in vacuum chamber
- cycled vacuum / air several times to make and break bubbles
- continued until almost all bubbles gone
- poured carefully over mask and put into oven
Preparing for the next steps
Making the mufluid circuits
In room 118
- 4 medium and 2 big circuits made were not destroyed by breaking the wafer
- Find cover slips and clean. They all fit on 18x24mm cs found in drawer by plasma machine
- Cut loose and peel off master
- punch holes for 1/16" tubes (which punch? test!)
- clean with tape
- plasma treat PDMS and CS
- bond
- connect tubes
Filling with water and gel
In laf bench in room 431, or maybe in 118 is OK?
It should be fairly sterile as well... If we wait long time between bonding the circuit and filling it the PDMS will become hydrophobic. The main idea of the procedure is that the PDMS should be slightly hydrophobic, but not too much to get the gel into the side chambers, but not to pass the column.
Idea for gel filling on/under microscope.
- collagen preparations need ice!!!! Ice is available in the chemistry department
- plug non-hydrogel inlets/outlets
- connect output to syringe with air to allow control of high or low pressure
- fill ungelled solution in pipette
- enter pipette in input tube and press solution through to outlet
- if solution does not enter lamella, increase pressure at outlet and keep pressing in solution, increase pressure until lamella are filled
- remove pipette and gently aspire gel solution from channel, leaving gel in lamellae
Questions:
- How to control inflow of such small amounts (1-10 ul)?
- A pipette is perhaps too brutal?
- Test with water in another circuit first?
- should I use the pressure control system?
- aspire 10 ul into tube with pipette
- couple tube to pressure control
- increase pressure until it flows through main channel
- increase pressure on opposite side to stop main flow and press into lamellae
- when lamella are filled
- either press harder with back pressure/ release pressure of pressure control
- or aspire on back end
- to fill main channel with air
- small volume syringe
- is probably precise enough?
- fill 1-2 cm of tube with solution. How do I do that? If this is 10 ul I can control this with a pipette or use our special syringes!
- syringe to aspire, to fill, then counter-pressure to fill lamella, then draw solution back into syringe
- fill main channel with DMEM media or PBS
Aliquoting
- Should be in final volume samples. How large? For gels in mufluid I now need max 2mm x 10mm x 0.1 mm = 2 mm^3 = 2 ul plus the entry volumes. Entry volumes: Ø 1 mm tube x 10 mm = 10 ul. This means that 30 ul is much more than enough!
- As a first trial, Ibid recommends using a 1.5 mg/ml Collagen I gel
- Pure or diluted
- Pure can be diluted to whatever later
- I only need 9 ul to make 30 ul ready collagen. How do I store such small amounts? Is that practical?
- Larger volumes will only end in waste...
- Diluted
- is a larger volume and easier to handle
- Dilute 99-100% acetic acid to 17.5 mM. How?
- Need DI water (new) for dilution
