Team:Freiburg/Protocols/iRIf Measurement

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iRIf - preperation and measuring (old setup/device)

How to prepare and conduct measuring with the iRIf device
remarks concerning the protocol (author, adapted from etc.)

material: chemicals, used kits, …
duration: … min

---

1. Add volume of added chemical (µL) (remarks, tips)
2. instructions time, speed, temperature (remarks, tips)

Preperation

• Wash flow chamber (rack for flow cell) properly with diH2O and ethanol (don't forget the tubes)

• Dry with nitrogen (don't forget the tubes)

• Wash flow cell properly with diH2O and ethanol; dry completely with nitrogen

• Put the flow cell onto the flow chamber; remember to:

1. do it with the right orientation (you have to see the actual flow cell; the two little tails at bottom)

1. fit the holes correctly (you have to see the opening of the flow tube)

1. rearrange flow cell (if necessary) to get rid of surface irregularities

• Put the clean, nitrogen-dried glass slide with the reactive side towards the flow cell

1. make sure it fits properly (does not lie on the edges of the plastik)

1. slightly push the glass on the flow cell (all but the actual chamber gets dark if it lies on correctly)

• Insert it at the magnetic holder at the iRIf device

Initialising

• Make sure PBS (10ml falcon) is in the first position of the racket

• Both tubes have to be outside the PBS: Start Init. protocol 1 → air is leeched into loading tube → remained fluid comes out

• Put 1 tube (yellow tagged) to the bottom of the PBS: Start Init. protocol 2 → loading tube is cleaned with PBS

• Put paper towel under the opening of the reagent tubes: Start Init. protocol 3 → reagent tubes are washed with PBS

    Fill 1,5 ml eppis with your reagents (300-500µl); Put them at the appropiate positions in the racket (3., 4.,... position in the racket
    
    Take care you dont loose the drop at the opening of the reagent tubes (insert tubes into eppis simultaneously) 

• Put second tube to the bottom of the PBS: Start Init. protocol 4 → filling tube (for flow chamber) is filled with PBS

Measuring

• Connect the ending of filling tube with the input tube of the flow chamber

• remove the cover of the lid; turn on electricity

• Start camera recording (choose appropiate folder for the output pictures)

• Start iRIf live view (by choosing the folder you just created for your pictures)

• Put the dimming rack over camera/flow chamber; check at live view for scattered light; rearrange rack if necessary

• Start an appropiate protocol to start the measurement

Washing the microfluidic device

* All steps are analogous to the initialisation of the system, but with diH2O and not with PBS!

• Make sure diH2O (10ml falcon) is in the first position of the racket

• Both tubes have to be outside the diH2O: Start Init. protocol 1 → air is leeched into loading tube → remained fluid comes out

• Put 1 tube (yellow tagged) to the bottom of the diH2O: Start Init. protocol 2 → loading tube is cleaned with diH2O

• Put paper towel under the opening of the reagent tubes: Start Init. protocol 3 → reagent tubes are washed with diH2O

• Put second tube to the bottom of the diH2O: Start Init. protocol 4 → filling tube (for flow chamber) is filled with diH2O

. .

protocols, irif