Spin free crosses_50758702

TheNessLab : Spin-free crosses

Created by Ahmed Raza Hasan, last modified on Dec 15, 2021

This method was developed to ensure highly efficient crosses that yielded as 'pure' a sample of recombinants at the other end. It's designed to ensure that parental cells are not lost during the cross (which is often the case in methods that involve spinning down parental liquid cultures) and also that the amount of parental cells that make it into the final batch of cells is minimized.

Disclaimer: Although the individual steps of this method are less involved than the alternative spin method, the overall timeframe of this method is nearly a full week longer due to the added gametogenesis steps.

Gametogenesis

1. Grow parental strains of interest in liquid culture until cultures are reasonably dense. I usually do this with 6 well plates left with shaking in the incubator for 3-4 days.
   • This only needs to be done once: after the cultures are sufficiently dense, the plate can sit on your bench and be used repeatedly for ~1 month and a bit.
2. While the cultures above are growing, make (or make sure you have) 1.5% agar plates for both regular and N-free media (Bold's or TAP). I'm going to assume you're using Bold's for the rest of this protocol.
3. For each parent, transfer about 50-70 uL of liquid culture to 1.5% Bold's plates, and use inoculating loops to spread the liquid culture all across the entire surface of each plate.
   • You technically just need one plate per parent, but I tend to do at least 3/parent each time I do this.
4. Leave agar plates in incubator until the plate has grown a thick lawn of cells. This typically takes about 3-4 days, or even 2-3 for some faster growing strains. I've usually left them in for 4 days just to maximize the amount of cells.
5. Once the cultures have sufficiently grown, use loops to transfer the lawns to N-free agar plates. Each time you do this, transfer the entire lawn but crowd the cells into about half the plate's surface area. I try to spread cells in a 'column' shape across the plate. Doing so will help ensure cells are nutrient-starved and gametes form.
   • This tip comes from a Chlamy workshop run in Geneva in 2006.
6. Leave N-free plates in light for 2 or more days. I try to cross on the 3rd day but I've gotten away with using cells that have only had two days in light. Avoid keeping the cells in light for more than 4 days though - they tend to discolour and mate less well.
   • If you want to store plates after this period, they can be removed from the incubator and stored in ambient light on your bench for up to 3-4 weeks (maybe more, but this is as long as I've had plates go so far and they've been fine)

Crossing

For this step, take 1 N-free plate for each parent of interest and transfer cells to N-free liquid media. Make sure to also leave some cells unmixed to serve as controls.

Other protocols I've found use distilled water here, but I haven't tried it. N-free has worked just fine for me - plus I figure using N-free makes absolutely damn sure all cells become gametes.

You're welcome to use whatever plate configuration you want for this as long as the cells are mixed somehow, but this is how I do it:

1. Obtain 24-well plate.

2. Fill wells with 2 mL N-free BM like so:

   1. N-					N-
      	N-	N-	N-	N-
      	N-	N-	N-	N-

3. Use a loop to scrape a small amount of cells into the top corner wells, which will serve as controls.

   • When doing this step, I tend to use the 24 well plate lid strategically to cover the other wells and make sure no cells accidentally fall off the loop inside.

4. Next, scrape equal amounts of the remaining lawn into the bottom 8 wells. I usually start by making sure each well has a bit added to it, so all wells have at least some cells in them. By the time I reach the last well, there's still lots left on the plate, and I transfer clumps a second time into the wells, being more careful to roughly add the same amount to each.

5. Use a 1000 uL pipette to mix cells up and down and break up cell clumps. I usually set the pipette to 750 uL here.

   • This helps mating but also makes adding equal amounts of the other parent to the wells in the next step easier.

6. Repeat with the other parent. Add small amounts of cells to the top right wells, and then add cells to the cross wells at the bottom. Make sure to add the controls first, since after that your loop will be touching cells from the other parent as you go back and forth between cross wells and your agar plate.

7. The final configuration should look like this:

   MT+ control					MT- control
   	cross well	cross well	cross well	cross well
   	cross well	cross well	cross well	cross well

8. Mix wells once again using a 1000 uL pipette until no large clumps are visible in any wells, best as you can. (Some strains like GB119 have clumps 'dissolve' very easily, while others like 2931 remain little 'flakes' no matter how much they're mixed)

9. Leave plate in light without shaking for 1-2 days.

   • Some strain combinations will mate quickly and form dense zygote mats in less than 24 hours, while others will get closer to that on day 2. I prefer leaving them for 2 days when possible because it also usually means less unmated cells are floating near the top, making the next step a little cleaner. 3 days is also fine but mats might get too crowded.

Dark maturation

1. Before collecting, make sure you have or have made sufficient agar plates. You’ll need 10 plates per cross. Label each plate with the strains being crossed, the date, and a well number (1-8). For less efficient crosses, 6 plates (4 for zygotes, 2 controls) can suffice.

2. (Optional) Obtain some liquid Bold’s media. You won’t need a ton (i.e. a thin sliver <5 mL in a tray should be more than enough).

3. Check 24-well plates for zygote mats under the scope. These should be visible with the naked eye, ideally. Check for swimming cells - there will be a few under the zygote mat even in seemingly efficient crosses, but if the cross went well there shouldn’t be a ton and should be hard to see under the mat if anything. Then, check the control plates - there should be no zygote mats whatsoever in those.

4. For each control well:

5. 1. Pull corresponding agar plate. I usually just do one per parent.
   2. (Optional) Pipette about 30 uL of Bold's liquid medium onto the plate
   3. Streak cells from both wells per parent onto the plate.

6. For each cross well:

7. 1. Pull corresponding agar plate
   2. (Optional) Pipette about 30 uL of Bold’s liquid media onto the plate, ideally in separate drops across the plate
   3. Use a pair of loops to disrupt the zygote mat and transfer it to the agar plates. This is best done under the scope.
      1. Two ways to do transfer the mat: either poke around the edges first to ‘loosen’ the mat before pulling it off, or split it in two down the middle before pulling out the separate chunks of it
      2. The mat/chunk will often curl in on itself as it’s pulled out of the well - this is normal and is dealt with in the next step
   4. Using the two loops, pull the zygote mat apart into smaller and smaller chunks. Liquid Bold's on the plate can help with this if you're struggling with it. This will break apart/flatten the clumped up zygotes (and ideally expose any unmated cells attached to them) - make sure all zygote clumps are ‘unfolded’ to expose unmated cells.
   5. Try to also scrape at edges of larger zygote chunks to break them apart - this will make life easier when breaking clumps post-chloroform.
   6. Place plate in dark immediately after streaking - I bring a sheet of foil with me into the hood, and as soon as I’m done streaking a plate I slide it underneath the foil

8. Once all the zygote mats as well as the controls have been transferred, wrap them in aluminum foil and leave on lab bench for ≥4 days. Make sure to label the foil with the strains crossed as well as the date.

Killing unmated cells (≥4 days later)

1. Make sure you have a set of autoclaved glass petri dishes.
2. In a biological safety cabinet, add a thin layer of chloroform to each dish, and cover with a glass lid.
3. Invert the agar plates onto the glass dish and cover with the glass lid. Leave for 45 seconds to a minute each.

Zygote germination + recombinant collection

1. Incubate the agar plates in light without shaking for ~2 days for the zygote to split into daughter cells and double a few times.

   • Zygote germination takes 15-20h (Aoyama 2009)
   • Sometimes, the centres of clumps of zygotes will still not have properly germinated - I've given it up to one more day for these to do their thing. You can usually tell since they look discoloured relative to the much greener recombinants.
   • The products of certain strains (i.e. any cross involving 2343) might germinate slower. 2343 F1s in particular tend to take 2-3 days to germinate at all, followed by another 1-2 days for doubling.

2. For each plate, scrape cells into 24-well plates containing 2 mL of liquid TAP per well. 

   • Do one well plate per cross - i.e. should have four well plates with 8 filled wells each

3. Leave the well plate in light for up to 12 hours, optionally with shaking. If shaking, use the timer on the VWR shaker to let the cells settle for the final hour or so. This step is quite flexible – I've gotten away with leaving them in light for just an hour or two.

   1. I've gotten by without shaking as well - it's usually only needed if the solution looks a little too transparent and you want to induce more growth.

4. For each cross, collect recombinants by spinning down cells into two 2 mL tubes. 

5. 1. First, for long-term storage, transfer about about 40-60 uL from the various wells onto a slant. Spread cells with loop and leave in light for 1-2 days to induce a bit more growth.
   2. If you want to PCR validate whether your final cells are indeed recombinants, transfer about 20-30 uL from each well to fresh wells of 2 mL regular Bold's in a separate plate and leave in light with shaking to grow for eventual extraction + PCR.
   3. Now that that's out the way - starting with the first well, pipette the ‘top’ ~1.5-1.9 mL of two wells into two 2 mL tubes respectively and spin down for ~30 sec. This should ideally only capture swimming recombinants, while dead cells will have settled at the bottom of the well and won't be brought into the tube. Discard supernatant.
   4. Repeat for the remaining 3 well pairs to concentrate recombinants into a single tube.

6. Leave 2 mL tubes in -80 freezer to prepare for eventual DNA extraction.

Document generated by Confluence on May 22, 2024 11:44

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