Objective

The long-range goal of this project is to quantify protein expression using two-electrode voltage clamp technique and fluorescence microscopy. We are expecting a linear correspondence between the two methods.

Methods

RNA Mixtures

Materials 

• 2.5 and 20 microliter micropipettes and pipette tips 

• 0.5 mL centrifuge tube 

• Nuclease-free water 

• OPRM1 with FLAG-tag RNA

• 1% CFTR 

• 10% CFTR with FLAG-tag RNA 

• 5% BAR

Procedure

10% OPRM1 0.1% CFTR 

1. Spray area with isopropyl alcohol. 

2. Put on gloves and use Eliminase. 

3. Obtain autoclaved 0.5 mL centrifuge tube. 

4. Add 8 microliters of nuclease-free water to tube

5. Pipette 1 microliter of OPRM1 with FLAG-tag into the tube 

6. Pipette 1 microliter 1% CFTR into tube

7. Store in -80C freezer until use

1% CFTR-FLAG 0.5% BAR

1. Spray area with isopropyl alcohol. 

2. Put on gloves and use Eliminase. 

3. Obtain autoclaved 0.5 mL centrifuge tube. 

4. Add 8 microliters of nuclease-free water to tube

5. Pipette 1 microliter of 10% CFTR with FLAG-tag into the tube 

6. Pipette 1 microliter 5% BAR into tube

7. Store in -80C freezer until use

Oocyte Injection

Materials

• Xenopus laevis oocytes 

• Binocular microscope

• Mesh-bottom petri dish

• 12-well plate

• Microinjector

• MBSH solution 

• MBSH + AB + AF solution 

• Oocyte incubator 

Procedure

1. Extract oocytes from Xenopus laevis then prep and sort oocytes. (see Protocol)

2. Spray area with isopropyl alcohol, put on gloves, and use Eliminase. 

3. Obtain mesh-bottomed petri dish and fill halfway with MBSH solution 

4. Transfer approximately 10 sorted oocytes into mesh-bottom petri dish using transfer pipette. Inspect oocytes through binocular microscope and ensure oocytes are healthy by looking for clear, symmetric border between the green and brown hemispheres. The oocytes should be similar in size and perfectly round, without any bulges or blemishes (i.e. discoloration, lines, etc.) 

5. Prep the microinjector and draw up approximately 1 microliter of RNA mixture. (see protocol) 

6. Line up the oocytes, impale, and inject 0.10 microliters of RNA. 

7. Obtain 12-well plate and fill well with MBSH + AB + AF solution. 

8. Transfer injected oocytes to well and label the well. 

9. Place in oocyte incubator.

Electrophysiology Data