Hi Brian,
Could you aliquot .5 mL PBS into at least 60 tubes? This will differ from a post a few days ago asking for 1mL (those are for Caroline), but both need to be done. Thanks!
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Articles posted by craigbateman
package awaits
There is a package(s) that needs to be picked up if you’re in today (Monday) from the main office. Might need a cart for it.
stuff to do
Hi Brian,
When you’re able could you:
1. make 2 L of PDA hard
2. put away and wash dishes
3. aliquot 1mL PBS to at least 30 microtubes
3. add vials to DB
4. autoclave old plates
old plates
Hi Brian,
More old plates ready for autoclaving
tubes and shipping
Hi Brian,
When you’re able, could you put yellow caps on three bags of cryo tubes?
The beetles that need to be entered into the database are in the freezer labeled “for Brian.” As a reminder, the host field is left blank, the capture type is “flying -> EtOH,” and the vial # is written on the top and side of the vial.
There is a package on a shelf above the printer that needs to be shipped. Instructions for sending it are on a page on top of the package.
Lactaphenol blue slide mount
- Place a drop of 70-95% ethanol on a microscope slide.
- Immerse the fungal material in the drop of alcohol.
- Add one or at most two drops of the lactophenol blue stain before the alcohol dries out.
- Holding the coverslip between forefinger and thumb, touch one edge of the drop of alcohol/stain with the coverslip edge, and lower gently to avoid air bubbles.
- Ring the edge of the coverslip with nail polish to prevent desiccation This way, the mount will last for days to months.
4th incubator
Could you bring up an incubator from the basement? It’s the same kind that as the ones next to the computer, and should be in a box somewhere around the main area. If you can’t find it, you may want to ask Adam where they were put.
Waste and Media
Hi Brian,
There are some old plates that need autoclaving next to the white board.
Also, we need 1L of quarter strength PDA and 1L of MEA (malt extract agar). The MEA will be in Jason’s lab, probably in a premade container like our PDA is. In the future, we’ll buy our own, but I want to try a test batch with some fungi first. When you find the container, could you write down the supplier/brand name and REF # and post it on here? Thanks!
Extraction from Xylosandrus compactus
- For one beetle, assemble thirteen 1.5 mL micro centrifuge tubes containing .5 mL of sterile PBS and label them as following:
(W)
(SW.1), (SW.01), (SW.001)
(MY.1), (MY.01), (MY.001) OR (G.1), (G.01), (G.001
(HP.1), (HP.01), (HP.001)
(A.1), (A.01), (A.001)
W=Water, SW=Surface Wash, MY=Mycangium, G=Gallery, HP=Head+ pronotum, A=Meso+metathorax+abdomen
- Add one drop of tween oil only to (SW.1).
- Wipe down microscope area with ethanol. Put the following items under UV sterilization for at least 10 minutes:
- Weak forceps, strong forceps, paraffin, minuten pins, 000 pins, and centrifuge tubes (see below). Open the cap of the tube with tween oil.
- Add the beetle to the tube with tween oil (SW.1). Vortex for 20 seconds at 2100 rpm.
- Remove the beetle from the tube with tween and add to a tube with sterile water or PBS (W). Vortex for 20 seconds at 2100 rpm.
- Transfer the beetle with sterile weak forceps to a dry kimwipe, and then onto the paraffin.
- Secure beetle with dorsal side up using minuten pins. Place four pins total in between the mesonotum and pronotum. Two of these pins should almost form a \/ shape in the parafilm, making a cradle for the beetle. The other two pins should form a /\ shape, holding the beetle down and preventing it from rotating.
- Insert a minuten pin into the anterior portion of the pronotum. Once inserted, decrease the angle the pin so that it is parallel to the paraffin and so that the pronotum of the beetle slides forward, revealing the mycangium. Secure this pin in the pronotum down with more pins to maintain its position.
- Using a sterile 000 pin, work the edge of the mycangial membrane to lift up the entire mycangium as a cohesive mass. The color is typically orangish or beige in compactus, and the texture looks like very fine sand or eggs. Transfer the entire mycangium into a centrifuge tube. This may take a few transfers.
- Using sterile scalpel, sever the beetle just anterior to the location of the mycangium. Transfer the head and pronotum into one tube, and the rest of the beetle into another tube.
Autoclaving stuff
Hi Brian,
Could you prepare 1L of PDA hard?
Also, in a separate autoclave cycle (solids) autoclave a beaker full of blue pestels and a beaker full of 1.5 mL microcent tubes. This task is a lower priority.
