Wednesday, 5 July 2017

In vivo shuffling via heteroduplex amplicons

Bluescreen transilluminator photo showing
different shades of green.
In a previous post I discuss the heteroduplicity of epPCR plasmids. Namely often colonies in a library will have bases that have two equal variants. The most likely cause being that the amplicons do not anneal perfect and that the transformed plasmids are actually heteroduplexes. These can either divide before being corrected or are corrected by mismatch repair.
I did a sneaky experiment to see if this could be used to make protocol to shuffle mutations between variants and got a positive result, but possibly not as effective as hoped.

Sunday, 23 April 2017

A note on Mutazyme and Manganese

Can Mutazyme be powered up?
Adding manganese works poorly, but stronger variants seem to be known.
(see also discussion about Mutazyme in Part I and II)

Tuesday, 11 April 2017

A simple hack for a phylogenetic Noah's ark dilemma

Ever had an endless list of bacterial names that needed a trim?
Ever see a tree where the bacterium chosen is not the famous one, but it's cousin? Or actually a tree where you don't recognise a single name?
The issue of picking bacteria from a list is what I call Noah's ark dilemma. This term is used generally for the biblical problem of the size of the boat required for all the animals in existence (except dinosaurs). Here I mean it picking the most meaningful bacteria from a list. In the past year, I have come to rely on a simple solution: Pubmed popularity.

Sunday, 26 February 2017

Peak height variation

Sequencing a plasmid pool containing a sequence with a randomised codon can reveal the frequencies of the bases are (Acevedo-Rocha et al., 2015 ).
The problem is that sequence traces are not consistent. Some peaks are bigger than others and beyond a certain point the traces get messy. So how does that affect the prediction of the base frequencies?

Friday, 6 January 2017

Top 5 useful facts about the MDS42 strain

MDS42, also known as the Blattner strain, is a strain made in 2006 by deleting 13% of the genome of E. coli K-12 MG1655. Unfortunately, as tools go, it rivals IKEA in cryptic instruction manuals.

Wednesday, 30 November 2016

Saturday, 12 November 2016

The heteroduplicity of error prone PCR plasmids

A mix of wt and
mutant...
In an error prone PCR the ep-aDNA is ligated onto a plasmid backbone and transformed. When assessing the diversity from a naïve plasmid pool, something odd is seen: some bases are mutated but not to saturation. This is often just dismissed or simply overlooked, but I suspect it is actually something interesting...