Team:Newcastle/15 July 2010

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(LacI BioBrick Construction)
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==Aims==
==Aims==
*To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
*To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
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*The aim of today's experiment is to '''screen for the lacI insert'''. The experiments that were carried out [[Team:Newcastle/16_July_2010#Protocol|yesterday]] are being repeated today as there were extra and unexpected bands on the image of the gel.
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==Materials==
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*[[Team:Newcastle/14_July_2010|Second round of Transformants]]
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*[[Team:Newcastle/12_July_2010|Plasmid extracts]]
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*Restriction enzymes ''Xba''1 aand ''Pst''1
==Protocol==
==Protocol==
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#Miniprep - the [[Team:Newcastle/Qiagen_Minipreps|miniprep]] protocol was followed.
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Single and Double digest of Plasmid extracts (single ''Xba''1)
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#Restriction digests - the [[Team:Newcastle/Restriction_digests|restriction digests]] protocol was followed to allow the length of the DNA to be checked using single and double digest.
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Run digests on a gel  
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#*Single digest with Pst1
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Overnight culture of second round of transformants
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#*double digests with Pst1 and Xba1
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#Gel electrophoresis - the [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] protocol was followed.
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#Set up liquid broth culture in 5 ml of LB - 1 colony from colonies 1-7. The protocol for [[Team:Newcastle/Growing_an_overnight_cultures| growing an overnight culture]] was followed.
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==Results==
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Revision as of 12:17, 10 August 2010

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Contents

LacI BioBrick Construction

Aims

  • To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

Protocol

Single and Double digest of Plasmid extracts (single Xba1) Run digests on a gel Overnight culture of second round of transformants

Results

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