EM - repeat hackytaq #30
Conversation
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@Starnite you can start reviewing as we go along. |
turns out there may be issues w/sequencing because of mixed product; this is just a check to make sure.
for future reference.
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| ## Interpretation | ||
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| One look at the gel and I realized what was going wrong. The melting temperature was incorrect. I had used 68ºC in this PCR round; in fact, it should have been 60ºC. |
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For all previous experiments we've been using Tm of 68, including the hacky-taq that I ran and the PCR we used to generate the low-mutation PB2 part 1 fragments for the cpec assembly.
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That's weird, hmmm...
Referencing back on this experiment, 68ºC yielded no PCR product for part 2, which is the hacky-taq PCR product for primer set EM-27/28, whereas at 60ºC the correct product shows up. According to that gradient PCR, EM-30/29 should be run at 68ºC.
Are you sure that the mytaq red PCR program was run using 68ºC, and not 60ºC? If you're not sure about it, may be good to start a new fork off master and update that note with a little bullet point stating that the Tm wasn't recorded down. (No worries that it wasn't recorded down that one time, these things happen, but it'll be important to note this down so that we remember for future reference.)
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@Starnite: I think I have some memory of what happened - that particular run, it was very likely 58ºC, not 68ºC. We chatted about it and decided to low-ball the annealing temperature, just to be conservative.
Nonetheless, the PCR conditions were not noted down, so I think it's best to add a note to the report indicating so.
After re-running the gel, obtained correct PCR bands.
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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. |
1. gel purification 2. sequencing
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ping @Starnite: please review. Summary of this set of experiments:
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@Starnite: This time round, I sent in all 10 µL of the PCR product for sequencing, so there's none left 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. |
@Starnite: Once again, I think gel purification is going to be necessary. If I get to start my own research group, I’m so totally getting a gel purification robot!
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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. |
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@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. |
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