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

DNA electrophoresis of restriction enzyme digested plasmids
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.

Procedure

Prepare the agarose gel by melting it in the microwave
Let the agarose cool down for a while
While the agarose cools down, prepare the plate to run the gel
Get the samples containing the DNA treated with restriction enzymes out of the fridge. The samples are as fellow:

     First set - treated with EcoRI

        Culture 2 (GFP(BBa_I13522)) - Lane 3 
        Culture 3 (RFP(BBa_J04450)) - Lane 4 
        Culture 4 (RFP(BBa_J04450)) - Lane 5 
        Culture 5 (RFP(BBa_J04450)) - Lane 6 
        Culture 6 (RFP(BBa_J04450)) - Lane 7 
        Culture 7 (RFP(BBa_J04450)) - Lane 8

     Second set - treated with EcoRI and PstI

        Culture 2 (GFP(BBa_I13522)) - Lane 9 
        Culture 3 (RFP(BBa_J04450)) - Lane 10 
        Culture 4 (RFP(BBa_J04450)) - Lane 11 
        Culture 5 (RFP(BBa_J04450)) - Lane 12 
        Culture 6 (RFP(BBa_J04450)) - Lane 13 
        Culture 7 (RFP(BBa_J04450)) - Lane 14

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. Both contain one restriction site for EcoRI and one for PstI. By just adding EcoRI we expected DNA to be linear whereas in tubes having both restriction enzymes we expected two pieces of DNA.

Add 1ul of loading buffer to each DNA sample and centrifuge for one minute.
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.