Team:Newcastle/12 July 2010

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=LacI BioBrick Construction=
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=Research=
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[[Image:P7120450.JPG|300px|thumb|right]]
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==Aims==
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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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Summary:
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We continued with our research on urease pathway in ''Bacillus subtilis'' 168.
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# Colony PCR
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# Streak plate
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# Minipreps
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==Materials==
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#Garnett J, Baumberg S, Stockley PG and Phillips SEV. (2007)."Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine". ''Acta crystallographica. Section F, Structural biology and crystallization communications''. 63(Pt 11). 918-21. International Union of Crystallography.
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* PCR reagents
 
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* LB plus ampicillin plates
 
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==Protocols==
 
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====Colony PCR====
 
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Colony PCR is a rapid way to check if the desired sequence is now present in the vector.
 
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7 colonies were selected for screening by colony PCR and Streak plating.
 
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[[Image:P7120427.JPG|200px|thumb|right]]
 
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A Positive control was performed to ensure that the PCR was working correctly
 
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The quantities for the PCR are as follows:
 
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Please refer to [[Team:Newcastle/PCR|PCR]]. The melting temperature, Tm, of this experiment is 54°C.
 
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'''Note''' We used 1 minute of extension time because the size of lacI is 1.4kb.
 
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====Results====
 
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No bands were observed from the seven test colonies, the positive control worked correctly.
 
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'''Note''' There are 2 possible reasons for red colonies formed:
 
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# partial digest - rejoins without insert
 
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# with insert 
 
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We need to tell the difference between the two.
 
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====Spread Plates====
 
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{|
 
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|-
 
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|[[Image:P7120408.JPG|thumb]]
 
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|[[Image:P7120409.JPG|thumb]]
 
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|[[Image:P7120418.JPG|thumb]]
 
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|}
 
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===Overnight culture===
 
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The seven colonies that grew on the plates were cultured overnight. The protocol for [[Team:Newcastle/Growing_an_overnight_cultures| growing an overnight culture]] was followed.
 
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==Tommorrow==
 
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We are going to take one of the colonies that have worked and possibly a whole plasmid prep.
 
{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Revision as of 19:20, 25 October 2010

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Research

We continued with our research on urease pathway in Bacillus subtilis 168.

  1. Garnett J, Baumberg S, Stockley PG and Phillips SEV. (2007)."Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine". Acta crystallographica. Section F, Structural biology and crystallization communications. 63(Pt 11). 918-21. International Union of Crystallography.


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