Team:Newcastle/Project/Labwork/Week1/Week3

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Introductory Lab Sessions Week 3

(14th July - 17th July 2009)

Lab session: 15th July

Experiment Recap

Two agar plates containing the 10 original cultures of transformed E.coli. Today's procedure will see the cultures in divisions labelled '2' and '3' cultured for a large plasmid prep

In the previous experiment, we had carried out DNA gel electrophoresis on six plasmids from our transformed E.coli cells - the six plasmids were each divided into two sets with the first set of six being treated with EcoRI alone and the second set being treated with EcoRI and PstI:
First set - treated with EcoRI

  • Culture 2 (GFP) - Lane 3
  • Culture 3 (RFP) - Lane 4
  • Culture 4 (RFP) - Lane 5
  • Culture 5 (RFP) - Lane 6
  • Culture 6 (RFP) - Lane 7
  • Culture 7 (RFP) - Lane 8


Second set - treated with EcoRI and PstI

  • Culture 2 (GFP) - Lane 9
  • Culture 3 (RFP) - Lane 10
  • Culture 4 (RFP) - Lane 11
  • Culture 5 (RFP) - Lane 12
  • Culture 6 (RFP) - Lane 13
  • Culture 7 (RFP) - Lane 14


After analysis of the gel photographs and comparison with the expected band sizes for the plasmids, plasmid backbones and BioBricks, we decided that cultures 2(GFP) and 3(RFP) were to be used for a larger scale plasmid prep.

To 2 tubes of sterile LB nutrient broth, a sample of cultures 2 and 3 were introduced. They were then placed into the shaking incubator overnight.

Introduction

Today's procedure is straightforward. Using aseptic technique, we are to innoculate 2 flasks containing LB nutrient broth with the 2 cultures grown overnight. Once carried out, these flasks will be incubated overnight and used in tomorrow's larger scale plasmid prep.

Procedure

Mathew pipetting 50ml LB into a flask



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