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Lab Work - 10/09/09

Metal Sensing Team

Introduction

Yesterday saw the Metal Sensing team attempt to transform DH5-alpha E.coli bacterial cells with two BioBricks (the cotC-GFP-smtA BioBrick and the kinA BioBrick) and plate them out on LB + kanamycin plates. The cotC-GFP-smtA BioBrick is part of the Metal Sensing team's sub-project and the kinA BioBrick will be used by the Stochastic Switch team's sub-project.

Today's exercise should see the Metal Sensing team inoculate LB medium with any transformant cultures which grow on these plates - if there are no cultures present then the transformations will be attempted a second time.

Observations

On all four plates there could be seen hundreds of transformant colonies - this was to be expected as the DNA used to transform the DH5-alpha cells was in pure form.

Procedure

There were two procedures carried out in response to these observations:

• Three colonies from each 200ul plate of transformants were used to inoculate 6 x 5ml LB+kanamycin (making a total of six tubes of inoculated 5ml LB + kanamycin). This is preparation for mini-preps.

• One colony from each of the 200ul plates (1 cotC-GFP-smtA transformed E.coli colony and 1 kinA transformed E.coli colony) was taken to inoculate 2 x 50ml LB + kanamycin media. This is preparation for midi-preps.

Preparations for Mini-preps

Three colonies were extracted from the 200ul cotC-GFP-smtA transformant cells plate and used, under aseptic conditions, to inoculate 3 x 5ml LB+kanamycin tubes. In the same way three colonies from the 200ul kinA transformant cells plate was used to inoculate 3 x 5ml LB+kanamycin tubes. These six tubes were then placed in the orbital shaking incubator at 37C for overnight growth.

Preparations for Midi-preps

When setting up 50ml LB cultures for midi-preps it is usual practice to inoculate the 50ml of LB with 50ul of LB culture taken from an overnight growth. However time was running short so we decided to take a risk and inoculate two flasks of 50ml LB + kanamycin directly from plates; one flask inoculated with 1 colony of kinA transformants and the second flask of 50ml LB+kanamycin inoculated with 1 colony of cotC-GFP-smtA transformants. These flasks were too placed in the orbital shaking incubator at 37C for overnight growth.

Stochastic Switch Team

Today we miniprepped sac and ara and carried out a test digest (EcoRI and PstI) on the samples in order to check if the insert hed ligated correctly. We also digested Hanny and James' sleB miniprep which needed to be cut with NheI and EcoRI. As it happened clear bands could not be seen for the ara and sleB lanes.