User Tools

Site Tools


wiki:protocols:foraging:protocol

Differences

This shows you the differences between two versions of the page.

Link to this comparison view

Both sides previous revision Previous revision
Next revision
Previous revision
wiki:protocols:foraging:protocol [2020/01/16 02:53]
mscholz
wiki:protocols:foraging:protocol [2020/05/11 08:21] (current)
nzjacic
Line 3: Line 3:
 ==== Strain ==== ==== Strain ====
  
-[[:wiki:documentation:strains|AML-283]]+[[:wiki:documentation:strains|GRU101]]
  
 ==== Microscope settings ==== ==== Microscope settings ====
  
-32.0x magnification\\ mRFP filter\\ Fluorescent bulb turned on and brightness set to 100%\\ 30fps+32.0x magnification\\ YFP filter\\ Fluorescent bulb turned on and brightness set to 100%\\ 30fps
  
  
 ==== pylon Viewer settings ==== ==== pylon Viewer settings ====
  
-Binning: 1\\ Gain: 5.0\\ 30,000 exposure+**For worms** 
 + 
 +Binning: 1\\ Gain: 0.0\\ 30,000 exposure 
 + 
 +**For lawns** 
 + 
 +Binning: 1\\ Gain: 20.0\\ 300,000 exposure
  
 **Saving recording**: **Saving recording**:
  
-As sequence of images, 1 Frame at a time, stop after 10 minutes+As sequence of images, 1 Frame at a time, stop after minutes
  
 +==== Conversion settings ====
 +
 +For 1x magnification 1px = 2.349 um.
  
 ===== Protocol ===== ===== Protocol =====
  
-**Syncing AML-283 adults**+**Preparing adults for synchronization**
  
-  * AML-283 takes ~66-67 hours until laying of first eggs - ideally looking for **65hr** adults.+  * To be able to record one strain per week: 
 +    * 5 worms generally make it to the lawn per video to get a decent sample size (100 worms) this means 20 recordings are needed 
 +    * 20 worms x 20 recordings =  
 +      * 400 worms in total = 
 +      * 200 worms per day = 
 +      * 100 worms during morning recordings and 100 worms during afternoon recordings 
 +    * That's a lot of worms!! 
 +  * To reach these numbers of worms requires some preparation 1 week in advanced 
 +    * Monday -  
 +      * Mutants: pick ideally 5x 1-2 day old adults onto a 10cm plate - repeat until you have 4x 10cm plates of adults - if you don't have enough for 4 plates or 5 adults per plate don't panic! - usually 1 plate of 5 adults is sufficient, but it's always better to be safe than sorry, especially if some plates grow contamination! 
 +      * GRU101 control: 1x 10cm plate with 3x adult worms is usually sufficient but make an extra in case of contamination 
 +    * Tuesday - repeat steps from Monday 
 +    * Thursday - take adults from Monday's plate and repeat the steps for both mutant and control strains 
 +    * Friday - repeat steps from Thursday 
 +  * By next week you should have plenty of egg-laying adults to choose from without the plate being starved! 
 +    * The benefit of doing this twice per week is that adults should only be about 1-2 days old when picking for synchronization i.e. Thursday worms are good for Monday syncing, Friday worms are good for Tuesday syncing! 
 + 
 +**Syncing GRU101 adults** 
 + 
 +  * GRU101 takes ~66-67 hours until laying of first eggs - ideally looking for **65hr** adults.
   * Set up one set of syncs Monday evening and one set of syncs Tuesday evening for data collection on Thursday and Friday morning-afternoon respectively.   * Set up one set of syncs Monday evening and one set of syncs Tuesday evening for data collection on Thursday and Friday morning-afternoon respectively.
   - Take two seeded plates - one is used as a lawn to remove unwanted worms while picking and the second is used for egg-laying.   - Take two seeded plates - one is used as a lawn to remove unwanted worms while picking and the second is used for egg-laying.
-  - From a healthy plate of AML-283, pick between 15-20 gravid hermaphrodites and allow to crawl on the lawn before transferring onto the egg-laying plate (ELP). Preferably pick adults of the same age i.e. day 1-2 adults. +  - From a healthy plate of GRU101, pick 20 gravid hermaphrodites and allow to crawl on the lawn before transferring onto the egg-laying plate (ELP). Preferably pick adults of the same age i.e. day 1-2 adults. 
-  - Allow the hermaphrodites to lay eggs for 2 hours.+  - Allow the hermaphrodites to lay eggs for 1 hour.
   - Remove all hermaphrodites from the ELP.   - Remove all hermaphrodites from the ELP.
   - Incubate at 20C.   - Incubate at 20C.
   - Check the progress of the ELP on day 2.   - Check the progress of the ELP on day 2.
   - Pre-egg laying adults should be grown after 65hrs.   - Pre-egg laying adults should be grown after 65hrs.
 +
 +**Syncing mutant adults with GRU101 as control**
 +
 +  * Each mutant strain has a slightly different time it develops into a pre-egg laying adult - to minimize stress all mutants should reach adulthood after incubating at 20C for 72 hours - to ensure that all strains and worms are roughly within the same developmental period during recording, there should be one synchronized batch in the morning (for morning recordings) and one synchronized batch in the afternoon (for afternoon recordings)
 +  * See above instructions for syncing
 +  * For GRU101 - also incubate for 72 hours along with the afternoon batch
  
 **Seeding testing plates** **Seeding testing plates**
  
   * 6cm NGM plates should be made fresh at the beginning of each week and put into 4C storage (if necessary) after drying overnight.   * 6cm NGM plates should be made fresh at the beginning of each week and put into 4C storage (if necessary) after drying overnight.
-  * Use 1.75ul OP50-GFP to seed a lawn exactly in the center of the 6cm plates. +  * OP50-RFP should be cultured before seeding: 
-  * OP50-GFP should be cultured fresh the night before seeding.+    * OP50-RFP requires gentamycin and strptomycin in at least 150ml LB: 
 +      * To make 10ug/ml gentamycin + 100ug/ml streptomycin in 200ml LB, add 40ul and 400ul of 50mg/ml gentamycin and streptomycin respectively when inoculating 
 +    * Take 1ml of culture and spin down at 13,000RMP for minute - remove supernatent and resuspend in 100ul LB with antibiotic resistance. 
 +      * 20mL of LB with 50mg/ml gentamycin and streptomycin should have 1mL aliquoted into eppendorfs and stored at -20C when not in use to preserve the antibiotics 
 +    * Due to the volume of LB necessary for culturing OP50-RFP, making one 10x per week and storing it overnight at 4C is acceptable. 
 +  * Dip the tip of a 100ul pipette tip into 10x OP50-RFP and gently touch onto the agar, ideally without poking a hole, to seed a lawn exactly in the center of the 6cm plates. 
 +  * Allow to dry for 24hrs. 
 + 
 +**Starving worms** 
 + 
 +  * Worms should be starved for 1 hour before recording - we've determined that this makes the distinction between foraging and feeding pumping more defined 
 +  * Take unseeded 6cm plates and pick as many worms as necessary (+ extra!!) for recordings - e.g. recording 6 videos during a morning session: 
 +    * 20 worms x 6 videos = 120 worms minimum + 80 worms extra = 200 worms 
 +    * 50 worms per plate = 4 plates of starved worms 
 +  * Be careful to pick plates with as few rips/bubbles in the agar as possible - worms CAN and WILL disappear into the holes/agar and you won't be able to find them again!
  
 **Setting up recording** **Setting up recording**
  
   - Put a 5ml drop of M9 0.5cm away from the lawn and opposite each other. Measurement should start from the center of the lawn outwards.   - Put a 5ml drop of M9 0.5cm away from the lawn and opposite each other. Measurement should start from the center of the lawn outwards.
-  - Pick 10 pre-egg laying hermaphrodites and immerse them in one drop of M9. Replenish the M9 if the drop is beginning to dry. Put another worms in the other drop.+  - Pick 10 pre-egg laying hermaphrodites and immerse them in one drop of M9. Replenish the M9 if the drop is beginning to dry. Put another 10 worms in the other drop.
   - Replenish both M9 drops before setting up the microscope camera. Place the plate under the microscope and find the focus of the pharynx as best as possible.   - Replenish both M9 drops before setting up the microscope camera. Place the plate under the microscope and find the focus of the pharynx as best as possible.
   - Center on the bacterial lawn, making sure the outside is completely visible.   - Center on the bacterial lawn, making sure the outside is completely visible.
   - Before recording, take a picture of the bacterial lawn.   - Before recording, take a picture of the bacterial lawn.
-  - Begin recording once the first pharynx comes into view.+  - Begin recording once the first pharynx comes into view and the pharynx is clearly focused.
  
 ===== Camera settings ===== ===== Camera settings =====
wiki/protocols/foraging/protocol.1579171993.txt.gz · Last modified: 2020/01/16 02:53 by mscholz

Bitnami