May 29, 2018
Flash Freeze Fruit Flies in Liquid Nitrogen
- 1Auburn University
Protocol Citation: Rita M. Graze 2018. Flash Freeze Fruit Flies in Liquid Nitrogen. protocols.io https://dx.doi.org/10.17504/protocols.io.nr3dd8n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: March 10, 2018
Last Modified: May 29, 2018
Protocol Integer ID: 10779
Abstract
A protocol for routine flash freezing of fruit fly samples prior to use in downstream protocols (e.g. RNA extraction).
Guidelines
Freezing should be done for a standard age or age range and within a 2 hour standardized window. Many genes are regulated according to the circadian rhythms and we want to minimize expression variation that results from the time of day.
Materials
MATERIALS
Drosophila VialsVwrCatalog #75813-160
Externally Threaded Self-Standing Cryovials with Silicone Washer Seal CapVwrCatalog #10018-734
2 Cryogenic box, with drain slots, with grids, 81 cell dividerVwrCatalog #89214-752
Before start
Make sure you have the following items: LN2, dewar (~2L), a large rectangular Styrofoam tub, colored timex tape and permanent markers.
Fly Sample Collection
Fly Sample Collection
When virgins are collected count how many are placed into each vial and write this on the outside of the vial.
Check virgin collection vials for larvae.
If no larvae present, consolidate by E# (cross identifier) into a single vial and write total number of flies for all vials corresponding to progeny from that cross of that type on the vial.
One Day to One Week Before Freezing
One Day to One Week Before Freezing
Create a spreadsheet that will record the information for all your samples and where they will be located in the freezer box and in the -80 freezer.
Before starting, check virgin collection vials for virginity a second time.
Pre-label freezer box with Experiment Title, Block # (if there are multiple blocks), Date, your name and email.
Pre-label Cryovials with V# (identifier for order vials are frozen in, starting @ 1), E # (cross identifier), sex, genotype, block #, and date; add the # of flies when you actually freeze. This information comes from your spreadsheet. You can use shorthand- but it should be explained longhand in your spreadsheet.
Day of the Freeze Before Starting
Day of the Freeze Before Starting
Put tape on bench to note the time tubes should be frozen (we allow flies 15 minutes to relax after being pounded through the funnel into the Cryovial).
Get liquid Nitrogen before starting at 3 p.m. (or the time you have chosen ... this will be fixed for all your experiments) and put in a rectangular Styrofoam tub that is big enough to hold two freezer boxes. Remember all flies must be frozen in a 2 hour window.
Freezing
Freezing
If you are freezing Cryovials of multiple genotypes, arrange vials so that no one factor is grouped by time (aka have a few of each genotype frozen at the same time). The easiest way to do this is to freeze all the vials that correspond to the first replicate, then the second replicate and so on.
For large freezes, we generally have 2 or more people seated in front of the racks of vials and freeze them from left to right front to back (with each person putting the flies in the vials in their section into cryotubes).
For freezes that do not use anesthetic- Select pre-labeled Cryovial and virgin vial, pound flies through funnel into Cryovial. Screw lid on loosely, but securely.
For freezes under CO2, knock-out flies, count them on the pad and sweep into the Cryovial. Screw lid on loosely, but securely.
Place Cryovial with flies in it on tape and note the time (we usually do this by grouping cryovials in 5 minute blocks) and in approx. 15 minutes place the cryovials in order by V number from left to right front to back directly into the freezer box that is sitting in liquid nitrogen. Make sure to do this quickly and that the vial is submerged. You can use tweezers to submerge the vial first to the side of the box and then put it in the right position in the box.
After you freeze them you can use the tweezers to rearrange them so that they are logical in terms of replicates and types or leave them in the freeze order. Whichever you do, you must make sure that your spreadsheet records where each cryovial is located in the box (A1 is the first slot, row A, column 1).