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📚 SGA & Libraries

SGA & Libraries​

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  • When planning an SGA and ordering plates, remember that there are plates that you can take from the general stock or from our “spare plates” stock. If you need plates from the general stock, take them in advance and put them in your fridge, otherwise they might no longer be there when you need them. Make sure to order more for the general stock if needed.
  • When ordering plates for SGA, order about 20% more than what is needed to account for pinning errors on the Singer.
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  • Keep all the plates from the SGA until you finish the whole process - you might need to go back and check patterns in the plates or start from the diploid selection.
  • Keep SPO plates outside the fridge after replicating to haploid selection - sporulation is generally very inefficient and you might have to return to the plates a few days later to get more spores. Additionally, you can also pin several times with different diameters (manually or using one of the Singer options) in the SPO plate to get a higher number of spores for each colony.
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  • If you are picking strains from libraries, then also ALWAYS streak to singles, since colonies can easily get contaminated with cells from close-by colonies. Perform a check PCR to ensure you are using the correct strain.
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  • When using the Singer, please alert Yeynit or Hadar immediately if anything is not working perfectly as this means that the Singer has to be serviced. For example, if the pad is not touching colonies = time to clean the pad head/check offset hasn't been changed.

  • While replicating on the Singer, if the corners of your plate do not replicate well, reduce the pressure of the pad gripper or use the mix option or elevate the edge of the plate with a small piece of tape (given that the agar is leveled).

  • For optimal Singer plates, if you pour your own plates, make sure not to pile them higher than 5 plates to let them dry as leveled as possible

  • When replicating from agar to liquid for imaging, there’s no need for liquid mix. It is also better to use lower pressure (30%), it gives a more homogenous replica.

  • Read the “SGA working protocol” before planning (all plans needed are detailed there). There is also a video (made by Ofir) that explains the basics of SGA, how libraries are made, and how to work with them properly. It’s in the lab shared folder under “protocols” (the only video)

  • If you want to make sure that your SGA will not be delayed, you can prepare and replicate 2 copies of SPO in advance (each of them in a different location) when moving from diploid selection. This way if the first copy does not grow well/gets contaminated on Haploid Selection after 5 days, you can use the second copy a few days later. Going back to a used SPO plate does not give good results….

  • When taking libraries from the general stock, make sure to take the correct copy (picking or replica). DO NOT take the ‘DO NOT TOUCH’ copy. If there’s a problem with the library, alert the person in charge of it.

  • If you are in charge of a library, you can use the “library cards” aid (the file is found in the Libraries folder, in “Aids and tools for library work") when refreshing it monthly. This aid creates a printable pattern of each plate and will allow you to easily track missing colonies and save them.

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-After you finish your SGA and before screening, make sure to fill the ‘Screen guide’ and do all the necessary checks. Send the file to Maya and Yeynit. You will have to get approval for starting the screen and a number for the screen from Yeynit. This is also relevant to screening small amounts of plates.

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-If you plan to change steps in the robot screen operation or you need a new protocol on the robot for screening - please notify Yeynit as soon as you start planning the SGA. Yeynit needs enough time to write the programs/ change them and run test runs before you can run your screen and this may take time.

  • If you need to thaw a single strain from a library you must get permission from Maya to do this. In general, taking out single strains is not allowed as it can cause them to disappear from a plate when the whole plate is thawed.

  • We reuse 384-well plate lids for screens: before placing ON plates in Eva incubator- clean the lids thoroughly with 70% ethanol (100% can be used for faster drying). Repeat the same cleaning in the next morning for the BK plate lids.