LabInfo/miscellaneous
From Kimmel Lab Wiki
Injection Equipment Help
Bonnie’s advice on pulling electrodes and injecting 1-cell embryos April 2007
Pressure injection equipment
- Pricing is probably out of date. Check with the companies for current pricing.
Stereo microscope suitable for injections: We got the Leica S4E zoom 0.63X-3X stereomicroscopes for the injection room. They were $1559 in 2002. We got the 10X oculars, one of them with a diopter adjustment. We got the 10446341 sub base with reflector and the 10446340 base and the 10446339 focus column. We also got the 10447153 frosted stage plate for live embryo observation. The part numbers are probably the Bartels and Stout numbers and maybe not the Leica numbers but B & S usually uses a permutation of the manufacturer's number as their number. Our rep is John Levin. The quote was QTL5918 from 12/2/02. Here's his email in case you have questions: johnl@bartelsandstout.com.
He has a new portable Leica stereoscope that I love. It's ~$800 if I remember right and it has built in fiber optics transmitted and epi illumination.... built right into the stand so no boxes for power supplies or epi. It's a teeny footprint, too and a low stand. The only drawback is that it comes with a clear stage plate. I've asked for this modification in the future.
Pressure injector
We get the other injection rig parts from Applied Scientific, 541-485-2284.
- MM33 Right Hand Micromanipulator with 1/2” clamp; Marzhauser #00-42-101-000. ~ $800 (old price, check website for current price)
- MMPI-/FS Foot switch with 8 foot cord ~$60 (old price, check website for current price)
- MPPI-2 ASI Pressure Injector. ~$895 (2008)
UPDATE 2008: new model is MPPI-3
- everyone who has had a chance to try it liked it at the UO.
- Date: Thu, 1 May 2008 13:14:41 -0700
- From: John Zemek <John@ASIimaging.com>
- Applied Scientific Instrumentation, Inc.
- John sent us the new MPPI-3 to try out. It got rave reviews for ease of use, efficiency and accuracy.
- Basically it is the same unit with a few minor changes. The operation is just like the old unit, but this one looks a bit different.
- The new unit will have the same price as the old unit: $895 for the basic unit without any accessories. ASI will be phasing out the old unit & it will be replaced by the new one.
Electrode holder for pressure injector from WPI 941-371-1003 MEH2S micro electrode holder for poke rig; 1.2 OD et w/ port ~$40
Electrode pulling and injecting
E212 storage jar for 1.2mm OD micropipets ~$35 1965 extra electrode gaskets for jar (might have a new part number, this is pretty old now) $15/6 gaskets
from FHC 800-326-2905 30-31-0 capillary tubing w/ Omega dot fiber; borosilicate; 1.2mmOD, 0.9ID, 100mm L (the kind that I like)
(from Sutter 415-883-0128 BF 120-69-10 electrode glass 1.2 OD, 0.9 ID)
You'll still need to scrounge up some tubing to connect the pressure injector to an air supply. You need some air pressure line to hook it up to a N2 cylinder or to building air. You should get a filter/regulator if you use building air and the pressure isn't too high there to be appropriate. Mike McHorse just goes to the local air components store with the parts in hand and finds what he needs. Pacific Rubber & Supply Corp at 690 Conger; 342-3350 We don't have part numbers from him. Here's what I've ordered in the past, though. Mike always had a huge roll of that stiff blue tubing so I've never actually bought that. Air Component Specialists 1-503-257-9848 MAR-1 air regulator; 0-100psi; 10-32 port, in line
and you'll need some Tygon tubing to connect the electrode holder to the pressure injector. There are various adaptors with the WPI kit so that you can use various size tubing.
Bonnie’s way of doing injections (that I sent to someone who I was helping troubleshoot some problems they were having):
I don't use Sutter, I use FHC cat#30-31-0, Boro 1.2 x 0/9mm ID w/ fiber electrode glass and made an laser electrode puller program to get sharp electrodes. For my program, I pulled lots and lots of electrodes and kept checking them at 100X. I monkey with the heat, duration and air parameters on the puller until I get a short very sharp tip that's not long and whippy. Mostly, I increase the heat compared to Sutter and increase the duration of the pull. Then for my injections, I just barely touch the end of the electrode with the flat side of a forceps while applying pressure with the foot pedal just until I see a tiny tiny drop of fluid coming out of the tip. Then I monkey with the duration dial on the injector until I get the right size bolus in the fish so that I only need 1 or maybe 2 pulses to get a nice size bolus in. I don't get tissue wicked up from the fish into the electrode and I don't get dribble of my dye or morpholino out of the electrode. If you have a solution that's dissolved in solvent for some reason, or in DMSO, it would be much more likely to dribble back out. I have my solution made in a physiological osmolality concentration of KCl usually. That is, in 0.1 KCl for large molecule dyes, like dextrans or 0.2M KCl or 1X Danieau for DNA type injections. I have injected drugs diluted in DMSO and not had dribble but it does tend to come back out of pipettes more easily.
My air pressure adjust on the top of my injector is between 30 and 40 psi on the dial.
I don't do the ramp tests on the electrode puller. Every once in a while, I have to make an adjustment in my parameters to keep getting my electrodes the way I like them. Mostly, it just takes a long time messing with a particular puller at a particular time and with the type of electrode capillary you prefer to get the result you want. A second puller of the same type in our department gave me a totally different result when I entered my program and used my electrode glass. I just look under the scope at the electrodes until I see them getting sharper at the very tip and then I keep varying that parameter incrementally until it no longer makes them sharper. Then I move on to the next parameter. There have been many books written on electrode pulling. It was faster for me to get the general idea and then just do it by trial and error. I would suggest taking the program that's already being used for sharp electrodes in your department, then try it with the FHC electrode glass. Try increasing the heat a bit at a time. Then the air, then the duration.
Micro injection molds for making agarose plates for lining up embryos for morpholino injections
The new number for Adaptive Science Tools is (773)-239-6133
inject@adaptivesciencetools.com
The address is:
Adaptive Science Tools 31 Gifford Drive Worcester MA 01606
http://www.adaptivesciencetools.com/ is the web site
We have ordered ones with troughs that have one slanted side and one vertical side to the trough for microinjection into zebrafish embryos
catalog number TU-1 for $50
Also, for photos, we have ones with small square holes for holding the embryos still
- catalog number PT-001 also for $50
old style blue silicon kit to make 1.2 % agar injection plate mold. CASTING MATERIALS STARTER SET from Micro-Mark
- (for molds to make silicone injection plates)
- catalogue number 82084
Morpholino folks
pmarcos@gene_tools.com
Paul Marcos is with GeneTools and has much technical expertise. Mention you're in the Kimmel Lab. He will undoubtedly make time to be very helpful to you, too. We’ve collaborated with them in beta testing several times and he’s great to work with.
http://www.gene-tools.com/ to order your MO on line after you’ve designed it.
