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

Goksel adding restriction enzymes EcoRI and PstI to the plasmid DNA

James Murray loading the wells within the agarose gel with enzyme treated DNA

Introduction

Today, DNA gel electrophoresis was carried out on the restriction digests of the plasmid DNA. Based on the observations from the gel electrophoresis, two cultures were then taken and used to innoculate LB broth.

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 follows:

- 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 to make the DNA heavier and centrifuge for one minute.
In the meantime, poor the agarose gel onto the tray, place the comb on the tray, and let the gel solidify.
After the centrifugation, place the DNA samples on ice
Prepare the lambda HindIII markers
Once the gel is ready, transfer the tray to the gel electrophoresis area
Fill with buffer
Gently get the comb off the gel
Start loading the lamba DNA ladder in Lane 1
Then load Lane 2 to 14
Finally load Lane 15 with the lamba DNA ladder
Set the voltage to 120 and run the gel electrophoresis
After 5mins, drop the voltage to 90V.

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.

Results

The photographs above show the DNA forming bands within the lanes of the agarose gel.

Next Stage

Mathew innoculating LB broth with the two desired cultures; culture 2 containing the GFP BioBrick and culture 3 containing the RFP BioBrick

It was decided that larger plasmid preps should be carried out on cultures 2 and 3 (containing GFP and RFP respectively). The E.coli cells from these cultures were taken from premade agar plates containing all ten cultures and used to innoculate 50ml of LB each. The E.coli within the LB were then grown overnight.

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.