Team:Newcastle/IntroductoryLabwork/14 July 2009

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Meetings Calendar

Introductory Lab Session: 14th July 2009

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.

20 – 21 June 2009 - Europe workshop (London)
23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
23 October Practice Presentation (Newcastle)
23 October T-shirts are ready
27 October Practice Presentation (Sunderland)
27 October Poster is ready
30 October – 2 November 2009 - Jamboree (Boston)

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[http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
[http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]

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 {{:Team:Newcastle/Left}}
 __NOTOC__
-===Lab session: 14th July===
+[[Image:Team Newcastle 2009 iGEM IntroductoryLabSessions.PNG|50px|right]]
+===Introductory Lab Session: 14th July 2009===
 [[Image:Team Newcastle iGem 2009 14-07-09 no 7.JPG|150px|thumb|right|Goksel adding restriction enzymes ''EcoRI'' and ''PstI'' to the plasmid DNA]]
 [[Image:Team Newcastle iGem 2009 14-07-09 no 13.JPG|200px|thumb|right|James Murray loading the wells within the agarose gel with enzyme treated DNA]]