So! Next round of mutation is coming up!
We looked at some protocols and decided that while we often use substitutions for various methods and materials this time we’re going to follow the instructions as closely as possible.
We’ll be using Agilent’s site-directed mutagenesis kit and going through all the control processes specified.
Normally we don’t do controls but since there have been some problems recently (growth, sequencing, possible mutations) I’m going to run through the procedures with the controls and do the double checking. The kit uses a pWhitescript control plasmid which we’ll use to transform into XL10-Gold competent E. coli cells. The XL10-Gold cells, if transformed correctly, should grow on a plate inoculated with ampicillin and look like small white-ish colonies. As an extra verification that transformation occurred as expected we can perform a test to see if the phenotype (blue in this case) is expressed on a media plate containing IPTG and X-gal. This might seem to be going overboard a little, but I think these are good methods to implement for this next round.
Before performing the mutagenesis I’ve streaked some LB plates with stocks from way back (verified non-contaminated with sequencing) to get template colonies ready. I made plates of 911T-C stored in DH5α and 911T-C LMG-194. Once I have colonies I’ll make 2 mL cultures in rich media broth and then mini-prep them to get naked DNA. We have a Nano-Drop 2000 in the lab which is great for determining concentrations. The nano-drop will give me the DNA concentration which I can use to calculate the how much material I should use for the mutation / PCR process.
I made NZY broth which we don’t normally use. It was a little tricky getting the pH correctly as the “magic wand” of our pH meter can be kind of wiley.
I hope my plates grow up nicely tonight!
Now to double check that all the pieces of the kit are present and then tomorrow – I’m going for it!