Team:Newcastle/12 July 2010

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'''Monday'''
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{{Team:Newcastle/mainbanner}}
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===Aims===
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=Research=
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[[Image:P7120450.JPG|300px|thumb|right]]
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The aim of todays Lab session was to perform the following(see summary) to isolate ''lacI''.
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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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# Miniprep PCR- of colonies that worked
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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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#Garnett J, Marincs F, Baumberg S, Stockley PG and Phillips SEV. (2008). "Structure and function of the arginine repressor-operator complex from Bacillus subtilis". ''Journal of molecular biology''. 379(2. 284-98.
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===Equipment===
 
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* PCR reagents
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{{Team:Newcastle/footer}}
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* Agar plates
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* Gloves
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* Wire loop
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===Colony PCR===
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Of the 11 plates with colonies grown 7 were used for colony PCR and Streak plating. Colony PCR is less accurate than the mini prep however results can be seen quicker whereas the miniprep has a 1 day wait. The colonies selected selected were satellite colonies because ampicillin degraded'''???'''
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7 Tubes were labelled 1-7 (+ a control)
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The quantities for the PCR are as follows:
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[[Image:P7120427.JPG|200px|thumb|right]]
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*1microlitre Template
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*0.25 microlitre GoTaq Polymerase
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*2.5 microlitre forward primer
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*2.5 microlitre reverse primer
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*1 microlitre dNTP (10molar stock)
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* 10.0 microlitre 5times GoTaq buffer
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* 32.75 microlitre deionised H20
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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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'''Note''' PCR is best when everything is kept on ice!
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'''Note''' Melting temperatures of primers is important and can have a big effect on the product formed (Temperature used see Thursday)
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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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|[[Image:P7120413.JPG|thumb]]
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|}
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===Tommorrow===
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We are going to take one of the colonies that have worked and possible a whole plasmid prep.
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Latest revision as of 19:32, 25 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

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.
  2. Garnett J, Marincs F, Baumberg S, Stockley PG and Phillips SEV. (2008). "Structure and function of the arginine repressor-operator complex from Bacillus subtilis". Journal of molecular biology. 379(2. 284-98.


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