injection.

here I am.
working in injection mode my problem is the continuous blockage of the injection needle (we are using 1mm capillars delivered together with the roboocyte). I tryed every time to increase the injection pressure to 3 bar but the blockage never has been removed.There is any treak to avoid this inconvenient? Furthermore the option " Iniection Shot" is always disabled. Why?
Wich is the suggested level of the solution in the wells during injection'

Hi Laura,
>working in injection mode my problem is the continuous
>blockage of the injection needle (we are using 1mm capillars
>delivered together with the roboocyte). I tryed every time
>to increase the injection pressure to 3 bar but the blockage
>never has been removed.There is any treak to avoid this
>inconvenient?

What do you want to inject? (cDNA / mRNA)?
How do you prepare it?

We use cDNA and the standard 1mm capillars and they work very well!

>Furthermore the option " Iniection Shot" is
>always disabled. Why?
In which menu / dialog?

>Wich is the suggested level of the solution in the wells
>during injection'
We use a TECAN Columbus Washer for plate filling. ~200µl (80-90% of the well)

Ciao,

Mike

Bayer Technology Services

Dear Laura

The needle tip is very fine and tiny. Therefore, blockage of the needle may occur in some cases, for example if the fluid in the tip dries out.

I would recommend to try the following if this occurs:
- Apply the highest available pressure (3 bar). If this does not help:
- Move the needle into fluid for a short moment(for example the liquid filled adjustment well without adjustment tool). If there are salt crystals, the blockage may be removed. If this does not help:
- Move the needle right onto the adjustment tool. It is very flexible and will not break, but it might be that the force is enough to remove a blockage.

If the blockage does always occur (which should of course not be the case), you should check your RNA/DNA solution, too. Maybe the concentration is too high, or there is too much salt, or too much cell debris in the preparation? Other users made there first tries with a dye staining the nucleus. Maybe this would help to get a bit of routine with the Roboocyte.

The disabled button: Switch to the Holding tabbed page and click Pressure first. Now the Injection Shot button is enabled. (It will be enabled on the Injection tabbed page too in the next software release.)

The recommended liquid volume for the wells is always the same: 200-250 µl. If you use the Tecan Plate Washer, you can fill the plates with this volume automatically before you plate the oocytes (see Roboocyte User Manual>Appendix>Preparation of Xenopus Oocytes)

Kind regards, Christine (MCS)

Many thanks to Christine and Mike.
Actuallty we realized that the RNA aliquote that we were using was prepared with an old procedure that induces the formation of crystals in the solution. They might be the cause of the blockage. We are now making a new preparation and the injection should run better next time.
The shot pressure botton was OK, I only had to press the holding pressure before

Now I'm starting with the first recording tests. So I'm coming back soon with new questions (I'm your nightmare). During the setting up I woukld like recycle the holders just changing the glass capillars, because you know, when you are at the first approach with a new system you use and breake a lot of disposable parts....Is that a bad idea?

Ciao

Dear Laura,

No, you are in no way a nightmare! On the contrary, we are happy that somebody uses this forum for discussing problems and (hopefully) feelings of success.

I think the crystals in your preparation are the cause of the blockage, too.

To your question: You mean, you like to replace the glass capillaries of the measuring heads? We do not recommend it, because it is a bit tricky. But you can try it if you like. You have to break out the capillaries (they are glued to the plastic part)and to rasp residues of the glue away (with a file). Then you are able to stick new capillaries into the slots. The distance between the tips is very important and should not exceed 500 µm (see also the User Manual).

If you are afraid of breaking the TEVC probe during adjustment, do not move the tips to near the adjustment device. This is not necessary because of the stepwise impalement procedure. Just make sure to have the tips centered. This is of course very important.

Christine (MCS)

I think the crystals in your preparation are the cause of the blockage, too.