AHHHHHHHHH I am angry with myself!!!!!!!!!!!!! again!!!!!!!!

This post might as well be an extension of the previous one. But it is just an attempt to organize my mind a little bit (sorry if it is poorly explain). I am trying to make competent cells of a Haemophilus influenzae strain with a antibiotic resistance marker (nov) in its chromosomal DNA in order to make a periplasmic experiment (explained a couple of posts ago). I picked from our collection a strain that had that antibiotic resistance mutation, but what I did not considered is that I picked and 86028NP strain with after reading Maughan and Redfield 2009 paper I realized that has lower transformation frequency than Rd strains. What implications might that have, well if I what to make a calibration curve with that strain, a lower transformation and uptake would give me a higher detection limit for quantifying DNA yield using that calibration curve ( see previous post).

So, I thought that I could use an RD strain on our collection, except that the only one that I could found was a Rd strain transformed with 86028NP DNA. This strain might work, but since it was transformed with entire genomic DNA I have no idea how the NP DNA has recombined in the Rd background. The best alternative seems to transform Rd with a PCR fragment of the nov resistance gene of our Map7 strain.  On the bright site, I could use this same PCR fragment as donor DNA to do a periplasmic extraction and assess the quality of the extraction in a agarose gel.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s