Team:Cambridge/Experiments/Initial Exercise Group A

From 2011.igem.org

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1. Template = B. subtilus genome.  Aiming to amplify ftsZ
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===1. ftsZ===
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Aim is to amplify ftsZ.
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{| border="1"
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|Template || B. subtilus genome
 +
|-
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|FWD Primer || ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT
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|-
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|REV Primer || agtgaacagctcttcgcctttacgGCCGCGTTTATTACGGTTTC
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|}
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Fwd : ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT
 
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Rev: agtgaacagctcttcgcctttacgGCCGCGTTTATTACGGTTTC
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===2. GFP side===
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Amplify fragment containing GFP coding sequence (RHS on diagram above) & part of vector plasmid
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{| border="1"
 +
|Template || Vector Plasmid. 
 +
|-
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|FWD Primer || AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact
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|-
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|REV Primer || provided
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|}
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2. Template = Plasmid. Amplify fragment containing GFP coding sequence (RHS on diagram above)
+
===3. Promoter side===
 +
Amplify fragment containing promoter (LHS on above)
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Fwd : AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact
+
{| border="1"
 +
|Template || Vector Plasmid. 
 +
|-
 +
|FWD Primer || provided
 +
|-
 +
|REV Primer || ATGTTTGTTTCGAACTCCAACATtttgaattcctcctttaattgg
 +
|} 
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Rev : provided
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===Returned Primers===
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3. Template = Plasmid.  Amplify fragment containing promoter (LHS on above)
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-
 
+
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Fwd : provided
+
-
 
+
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Rev: ATGTTTGTTTCGAACTCCAACATtttgaattcctcctttaattgg
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===Primers===
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[[File:CAM HJCA PRIMERS.JPG | thumb | 850px | center | The Technical datasheet we received]]
[[File:CAM HJCA PRIMERS.JPG | thumb | 850px | center | The Technical datasheet we received]]
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Revision as of 13:44, 7 July 2011

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Contents

Initial Exercise: Cat, Jonathan, Haydn and Ai

As a 'warm-up' exercise to acquaint the group with molecular biological laboratory techniques, three mini-teams were tasked with creating an interesting GFP fusion. Group A decided that visualing ftsZ in real time, in vivo would be rather cool.\

Ftsz was first identified in a mutant screen in 1980 <ref>(Lutkenhaus, Wolf-Watz and Donachie)</ref> as a gene recquired for bacterial cytokinesis (cell division).

Notes

Template:Reflist

Primer Design

The plasmid vector we were supplied with contains a strong promoter upstream of a sfGFP coding sequence. Our fusion design relies on amplifying the ftsZ coding region from Bacillus and creating regions of overlap between this and the GFP coding sequence in the plasmid, in order to create the gene fusion.

The desired end product is shown below, with the plasmid in lowercase and the ftsZ coding region insert in upper case. 

ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT-----AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact
ggttaatttcctccttaagtttTACAACCTCAAGCTTTGTTTGTA-----TCTTTGGCATTATTTGCGCCGgcatttccgcttctcgacaagtga


1. ftsZ

Aim is to amplify ftsZ.

Template B. subtilus genome
FWD Primer ccaattaaaggaggaattcaaaATGTTGGAGTTCGAAACAAACAT
REV Primer agtgaacagctcttcgcctttacgGCCGCGTTTATTACGGTTTC


2. GFP side

Amplify fragment containing GFP coding sequence (RHS on diagram above) & part of vector plasmid

Template Vector Plasmid.
FWD Primer AGAAACCGTAATAAACGCGGCcgtaaaggcgaagagctgttcact
REV Primer provided


3. Promoter side

Amplify fragment containing promoter (LHS on above)

Template Vector Plasmid.
FWD Primer provided
REV Primer ATGTTTGTTTCGAACTCCAACATtttgaattcctcctttaattgg

Returned Primers

The Technical datasheet we received



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