Guelph/2 October 2008

PCR and Culture -- Verification of Transformation

Present today is me and Jenn Vo-- Dave was here to advise then he went to class.

Dave's left us with a few procedures that we can do. The first f which is a PCR to just diagnose whether or not a transformation worked -- I'm not 100% clear on which colonies we are doing but we're basically checking to see if the inserted sequences are there. We will be working on four colonies selected near the centre of the culture plate.

This PCR is to be done on whole living cells-- one swab of cells for each of the eight selected colonies, so we're going to run a boil program on the thermal cycler to lyse the cells and liberate the plasmids to sequence.

Simultaneously, the cells from each of the swabs will be cultured so we can visually inspect whether we have the right cells. Culturing is done in 10mL tubes suspended in LB culture fluid instead of on plates. Cells will rest overnight in the shakers.

Notes in Sequence...

* We borrowed some sterile LB from stock. * Carbinicillin was added at a concentration of 1 uL per mL. * Colony 1 in the LB fluid was contaminated by Ed's fingers -- Sorry. * All other mixtures OK, all PCR colonies OK including colony 1. * Procedure paused -- Need Dave to demonstrate thermal cycler operation-- it's a model I haven't used before.

Master Mix and Thermal Cycler

Jenn took over for a while after Dave returned, and produced the first set of PCR ready mixes. I'm producing the second. I've distributed the master mix of reagents into the eight PCR tubes-- 1uL of template DNA is mixed in and 15uL of oil is added on top. I slipped while loading sample number eight so there may be contamination in there. The last volumes of template DNA and mineral oil mentioned came from Fermentas' online PCR protocol. The thermal cycler was started at 13:24.

- Eddie Ma

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Heat shock procedure with competent E. coli cells

Today, I'll be transforming E. coli with two plasmids ("2ox1 and 20ox02"). I'm using 5uL of each plasmid and adding it to two stocks of competent cells each. Total volume is roughly 1mL.

The cells have been heat shocked at 42ºC for 60 seconds, iced for 120 seconds and treated with 200uL of SOC each tube for recovery in shaking chamber. Total volume of SOC added before recovery period in shaking chamber is 1200uL-- the added 1mL is to allow the bacterial culture more room to grow.

There was roughly a five minute delay between the time the first SOC was added and the time when the cells were actually introduced to the recovery apparatus (had to find the right vessels to hold the cultures, then transfer the cells over). There is now a two hour waiting period before the next step of plating the cultures.

- Eddie Ma