Multichannel Systems Roboinject Manual - Manual (Page 73)

Troubleshooting
69
Injection needle gets blocked during injection
If the injection needle is fine when you start the injection, but gets clogged during the injection
sequence, impurities in the DNA / RNA sample can still be the cause, because the particles may sink
down slowly. Check the possible causes for the problem "Injection needle is blocked from inside"
as well as the following.
Possible causes:
? Contaminations in the cDNA / mRNA sample slowly sink down during injection and cause
the needle to clog.
Inject a well plate without oocytes, for example a well plate filled with mineral oil. Check the
injected volume with a microscope. If the problem persists, the cDNA / mRNA sample itself causes
the trouble. Please see preceding paragraphs "Injection needle is blocked from inside" for further
advice.
No or Low Expression
You should expect an expression rate of close to 100 percent for mRNA injection and a success
rate of up to 70 percent when injecting cDNA, depending on the receptor and the oocytes,
of course.
Please make sure that the DNA / RNA sample you use has been sufficiently tested and has a good
expression performance. Make sure that the oocytes show a good general performance as well.
Oocytes are not hit during injection
If the injection needle does not clog, but the oocytes do not show a high expression rate,
you should check whether the oocytes are hit during injection. Please perform a test injection
with the dye Trypan Blue to analyze the situation further (see "Strategies for a Successful
Injection" in the "cDNA / mRNA Injection" section).
Possible causes:
? The wrong well plate type has been used.
Make sure you use a well plate of the type that you defined in the General Options dialog box.
Check the "Sources of Supply" if you are not sure about the recommended product.
? The well plates are warped.
Well plates have to be very flat to make sure that the oocytes are centered and hit properly.
Check each plate before use. Do not use warped or sterile-packed plates (as they are known
to have problems with warping due to the sterilization process). Do not autoclave or sterilize
plates by heat.
? The alignment has not been performed properly.
Repeat the alignment and make sure that you see the needle clearly and three-dimensionally
under the microscope. The fine tip of the needle should be positioned inside the groove in the
middle of the crosshairs. Practice the alignment process without actually injecting to get used
to the procedure. Let another person aid you if you are not sure whether the tip of the needle
is really centered on the alignment device.
? If the alignment is fine, the impalement and injection depth have to be optimized. Generally,
the default values (300 / 550 μm for cDNA and 650 / 550 μm for mRNA) are fine for all oocytes.
But you may want to optimize them if your expression rate stays consistently below 80 percent,
even for a good receptor.
Modify the injection distance values until you successfully hit the oocytes. Start with the standard
values (300 μm for DNA and 650 μm for RNA) and lower it in steps of 50 μm.