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ericmjl / protein-systematic-characterization

All our protocols, data, analysis, and papers related to this project are stored here.
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EM - repeat hackytaq #30

Closed ericmjl closed 7 years ago

ericmjl commented 7 years ago

@Starnite you can start reviewing as we go along.

ericmjl commented 7 years ago

Current status: just sent in PCR products for sequencing. We will get back the sequencing results tomorrow morning. I hope it'll be correct this time round.

ericmjl commented 7 years ago

ping @Starnite: please review.

Summary of this set of experiments:

  1. definitely want to gel purify before sequencing. All of the samples had good CRL scores (see Genewiz screenshot), much better than my previous two attempts, and moderately better than your first experimental run.
  2. I can see why 0.5 mM Mn2+ is used, though I think this deserves one more replication to be sure.
ericmjl commented 7 years ago

@Starnite: This time round, I sent in all 10 µL of the PCR product for sequencing, so there's none left ☹️. That's because the DNA concentrations were low enough necessitating all of the eluted product being sent in.

If you look at the temp-hackytaq notebook, you'll notice that the replication run had much fewer mutations than before. Given that, I'm not quite yet confident in the number of mutations that will show up, so I think we'll need a replication run of the hackytaq PCR reactions to get a more reliable estimate of which concentration of Mn2+ to use.

ericmjl commented 7 years ago

ping @Starnite: let's not merge this in yet. I think it's a nice mini-experiment that can go on the side whenever there's downtime, and I can see the data coming in handy with the Bayesian analysis paper.

ericmjl commented 7 years ago

@Starnite I think I'd like to merge in.

After chatting with Stuart (BioMicroCenter), I think we can take this off the priority list. It was a fun experiment to have run, nonetheless.