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Contents 1 Introductory Lab Sessions Week 3 1.1 The third week initial planning, 14th July - 16th July 1.1.1 Lab session: 15th July 1.1.1.1 Experiment Recap 1.1.1.2 Introduction 1.1.1.3 Procedure

Introductory Lab Sessions Week 3

(14th July - 17th July 2009)

The third week initial planning, 14th July - 16th July

• Tuesday

1. DNA electrophoresis of restriction enzyme digested plasmids
2. Innoculate LB with two selected cultures (decision based on DNA gel electrophoresis results)

• Wednesday
  • Innoculate 2 flasks of LB with the two selected cultures in each one - leave overnight for 16 hours.

• Thurday
  • Prepare large plasmid preps from both cultures
  • Carry out DNA gel electrophoresis

Lab session: 15th July

Experiment Recap

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

• Prepare 2 sterile flasks and to each of them, add 50ml of sterile LB.

Remember to use aseptic technique throughout, i.e. flame the lids of the flask and the LB bottle with a Bunsen burner before and after transferring liquids

• Label the flasks appropriately. To one flask, pipette 100 microlitres of the first culture and to the second flask, pipette 100 microlitres of the second culture. (In this case, they are cultures 2 and 3 - GFP and RFP transformed E.coli respectively)

• Place flasks in shaking incubator. To ensure that the right amount of cells grow in the media for the plasmid prep, the incubation period should be limited to 16 hours.