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Introductory Lab Sessions Week 3

(14th July - 17th July 2009)

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

Prof. Wipat demonstrating lab techniques

• 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: 14th July

Introduction

Today, we carried out DNA gel electrophoresis. The steps we followed are below:

Procedure

• We prepared the gel by melting it in microwave and cooling it down for a while.
• We got the samples containing dna + invertases out of the fridge. Six of them them had GFP + EcoRI and the other six had RFP + EcoRI + PstI. By just adding EcoRI we expexted DNA to be linear whereas in tubes having both invertases we expected two pieces of DNA. We placed 1ul of loading buffer to sink the DNA and centrifuged for one minute. Plasmid with GFP(BBa_I13522) is resistant to AmpR and KanR and the other one with RFP (BBa_J04450) is resistant to AmpR and TetR.
• We first loaded the standard DNA ladder. After the ladder, we loaded the other samples to the wells and run the electrophoresis. We set the voltage to 120 initially and then dropped to 90V afterwards. Because the DNA is negatively charged, this voltage moved the DNAs on the gel towards the positive side. We then looked at the DNAs on the gel by ultraviolet light.

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

• 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.