Notes for demonstrators

Day 1

This session takes the full three hours (so encourage people to get started)  but there is quite a bit of downtime. There are some extra activities for them to do (I will print out answers for these).

1. Annotating the plasmid. They need to understand what all the bits on the plasmid do. Please feel free to explain anything they don’t get.
2. Predicting results for next time. Happy for you to help with this but maybe don’t tell them the answers until next time, when they can compare their predictions to their actual results. I think this is more helpful.

Common things that go wrong:

• There are a few different temperatures today and someone always gets confused – make sure they’re using the right hot blocks at the right time!
• There are two different concentrations of lithium acetate (that look rather similar!) Someone always gets these mixed up. They might need to start again, depending on what went wrong and when (but we have limited time, so we might need to be creative):
  • They can possibly shorten some incubation times:
    • Lithium acetate incubation from 30 mins to 20 mins
    • 1 hour recovery to 45 minutes
    • These might reduce efficiency but, if they get no transformants, they can borrow plates next time.
  • Or they can potentially borrow cells from a neighbour (they have more cells than they need to transform at the end).

Day 2

Things to remember today:

• If anyone has no colonies, they can share with neighbours/friends (assuming there are enough colonies). If this isn’t possible, there will be rescue plates. (These are a bit of a fudge! They’re a mix of possible genotypes, plated out. Sometimes, this gives odd results on day 3 so best to share with neighbours if possible).
• Transformation efficiency – they should use the mass of pBH750 only (this is the plasmid being selected for). Check they are not using the mass of both plasmids for transformation 1. Maths should be quite easy! 1 ug of plasmid was transformed, so efficiency = number of colonies ÷ 1.
• They will have lots of tubes and lots of plates with lots of samples. Labelling is really important today. The set up of the screen usually needs explaining to a few people as there is just a lot going on, so please be sure you understand it too.
• Plates of controls (+ and -) are needed in the afternoon. Don’t throw them away in the morning.
• Guide RNA design – make sure you are comfortable with explaining this as they do need to understand how to do it. They found this complicated last year (I have updated it based on comments but it’s always possible I’ve changed it for the worse!) I would particularly like them to be able to deal with reverse-complementing DNA; knowing which end of the RNA is the scaffold and which is the gRNA; whether the gRNA binds top/bottom strand. It would be a good idea to work through this exercise yourselves before the session. Yes, they can just get this info from the online programme but that programme won’t be there for them in, for example, an exam.

Day 3

• There is very little lab work today – just looking at plates.
  • The met screen is not black and white and usually generates a lot of questions. It is affected by how many cells were in the original suspension.
  • I would recommend they look at positive and negative controls first to make sure they understand what Met+ and Met- phenotypes look like.
  • Second, check the +met plates to make sure they have comparable growth.
  • Third, look at the -met plates.
  • It’s fine for them to just make a decision (and it’s fine if it’s the wrong decision! We would genotype colonies anyway).
• Primer design – also usually tricky
  • When confirming GFP integration at the right place, they should avoid using primers in the homologous regions (as these are also in the GFP cassette).
  • It’s a good idea to avoid very large PCR products (anything that would be >2 kb or so). But I’m not going to be strict about this.
  • I would like them to be able to
    • Plan where primers are.
    • Know what orientation the primer should be.
    • Know what different primer pairs will tell you about the genome.
    • Design primers that will confirm integration at the right place.
  • I would like them to have a go at writing out the sequence of at least one primer pair. More if they want! Check that they reverse complement any reverse primers that they design.

Handouts

Day 1: plasmid annotation exercise printout: 22-23 BIS205 practical – plasmid printout

Day 2: sgRNA design exercise printout: sgRNA design (Lt)

Day 3: PCR primer design exercise printout: 22-23 BIS205 practical – PCR primer design