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I need to clone 96 different inserts into the same backbone in arrayed format. I am planning to perform Gibson assembly reactions in a 96 well plate, and then do chemical transformations in a 96 well format.

However, I am not sure how to plate 96 well transformations in the most efficient way. I could just plate them one by one onto petri dishes, dividing each petri dish into 4 sectors. However, I suppose there could be a better way of doing it. Any suggestions?

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    $\begingroup$ You could plate them in 6-well or 12-well plates, so you don't have to juggle all the Petri dishes. $\endgroup$ – MattDMo Oct 15 '20 at 1:24
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    $\begingroup$ If you want to plate them as usual you can use glass beads instead of a spreader to spread them. $\endgroup$ – GaelC Mar 31 at 22:46
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I think your approach very much depends on how essential it is that you get discrete single colonies, and if this is a one off or a regular process. To preface, similar to what Maximilian Press said, using a single welled plate like a nunc omnitray would be your best bet.

The main consideration you need to take into account for high-throughput transformations is the transformation efficiency (TrE). Naturally in a big petri dish, 100 µL of a single 1:10 or 1:100 dilution of your recovery culture can bring out nice discrete colonies. When you start reducing surface area and volumes, the chance of discrete colonies obviously diminish.

The best way I've found is to do a test transformation using the backbone of your choice and gauge the average TrE over a few runs to hone in on a 'one size fits all' dilution/volume, then apply that to your full high-throughput run. Assuming your inserts are of similar size, you should see a relatively similar TrE between them (bar edge cases). Another option would be set up three 96-well plates and do three separate dilutions, 1:10, 1:100 and 1:200 at 100 µL a pop so you at least get some fudge factor.

In terms of the chemical transformation itself, doing them in a 96 well plate format will guarantee lower TrE simply due to the characteristics of the plate itself. Thicker plastic and sub-optimal heating (if you aren't using a thermocycler and PCR plate) requires a slightly longer heat shock, and recovery in media requires much more vigorous shaking due to the mixing characteristics within the wells.

If you don't care about plating out in 96 well plates, like Maximillian Press said, use a single welled plate and format it in a 96/48 well fashion. Will save a massive headache when pouring. Just make sure you leave it to dry to prevent any running culture.

In terms of actually growing the colonies in a 96 well plate, if you're making the agar yourself I'd recommend adding an extra 0.5% agarose to thicken it up (I doubt you'll be using one, but make sure your plate is non-tissue culture treated). Wait for it to cool just above its solidifying temperature (around 45 °C in my experience) and then transfer out using a pipette or low volume serological pipette to reduce the meniscus formation. If the agar forms a high angled meniscus, your colonies may form smears and will not be discrete. Leave the plate out to dry as well, colonies may also smear from the agar being moist.

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    $\begingroup$ TBC, I was talking about plating on a flat plate, not 96-wells! It has the same shape and size as a 96-well plate but is otherwise a flat bottomed plate. The similar format makes it easier to organize and avoid errors, but pouring 96 wells sounds like a huge headache. $\endgroup$ – Maximilian Press Apr 1 at 10:51
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    $\begingroup$ Ahh yes I was agreeing with you, just mix up in what I was calling them! I agree, using actual 96 wells is a real pain. $\endgroup$ – Matt Apr 1 at 11:40
  • $\begingroup$ gotcha, cool, seems like we're on the same page. $\endgroup$ – Maximilian Press Apr 1 at 15:48
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I would suggest pouring solid media selection plates of something like this, that have the same shape and size as the 96-well plate. You can then directly spot your transformations from their wells onto this plate, which has a similar format. You will not be able to pick colonies per se (you will just have a confluent spot of cells that can grow on the selection), but if you are just interested in the constructs it would save a lot of labor and potential confusion.

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    $\begingroup$ An alternative to the confluent spot could be to do spot plating from dilutions as it is done in survival experiment. This would allow the recovery of individual colonies. $\endgroup$ – GaelC Mar 31 at 22:36
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    $\begingroup$ @GaelC a good point, though I worry about transformation efficiency being high enough to reliably recover colonies from dilutions. Maybe do a 1:10 dilution series and plate each dilution, to account for variation between reactions? $\endgroup$ – Maximilian Press Mar 31 at 22:45
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    $\begingroup$ Yes, for the first time I would do that and see if 2 or 3 dilutions points could be enough for the next time. $\endgroup$ – GaelC Mar 31 at 22:53

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