Team:Groningen/7 June 2010

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'''Week 23'''
'''Week 23'''
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'''Arend Jan'''
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All the PCR product was used up so a new PCR was performed.
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<pre>Component         µl Final concentration
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Primer pNZ89bbs-for1 5 300nM
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Primer pNZ89bbs-rev1 5 300nM
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10x pfu buffer(+MgSO4) 5 1x
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dNTP’s            2 200µM
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Template         0.5 ~14ng
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Pfu polymerase    1 2.5u
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MQ                 31.5
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- 94°C, 3’
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- 94°C,  30’’
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25X - 50°C,  30’’
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- 72°C,  5’
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- 72°C,  10’ </pre>
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[[Image:08-06-10gn.jpg|200px|thumb|left|Last time in bad quality, I promise]]
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<br style="clear: both" />
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Using 10x pfu buffer + MgSO4 (instead of adding MgCl2) seems to decrease smear. Also, increasing the elongation time to 5min seems to give more product. Product was cleaned (Roche kit)and digested with BamHI and ligated.
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<pre>- 22ul plasmid
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- 2.5ul buffer G
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- 0.5ul BamHI
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- 10ul/20ul DNA (~100ng/~200ng)
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- 5ul T4 ligase buffer
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- 5ul T4 ligase
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-30ul/20ul MQ </pre>
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25ul of ligation mixture was used for transforming 100ul compentent cells. Good amount of colonies. Colonies were picked, grown overnight, and minipreped using the High Pure Plasmid Isolation kit from Roche.
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Long weekend including Paris workshop.
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<br>
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'''David'''
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<br>
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Growing streptomyces for purification of the chaplin proteins.
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<br>
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Protocol: [[GrowingStreptomycesGR]]
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<br>
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{{Team:Groningen/Footer}}

Latest revision as of 19:28, 24 October 2010

iGEM Groningen 2010

Hydrophobofilm
pushing coatings into a greener future

Week 23



Arend Jan


All the PCR product was used up so a new PCR was performed.


Component	        µl	Final concentration
Primer pNZ89bbs-for1	5	300nM
Primer pNZ89bbs-rev1	5	300nM
10x pfu buffer(+MgSO4)	5	1x
dNTP’s             	2	200µM
Template	        0.5	~14ng
Pfu polymerase    	1	2.5u
MQ	                31.5	

- 94°C,	 3’
	- 94°C,  30’’
25X	- 50°C,  30’’
	- 72°C,  5’
- 72°C,  10’ 


Last time in bad quality, I promise



Using 10x pfu buffer + MgSO4 (instead of adding MgCl2) seems to decrease smear. Also, increasing the elongation time to 5min seems to give more product. Product was cleaned (Roche kit)and digested with BamHI and ligated.


- 22ul plasmid 
- 2.5ul buffer G
- 0.5ul BamHI

- 10ul/20ul DNA (~100ng/~200ng)
- 5ul T4 ligase buffer
- 5ul T4 ligase
-30ul/20ul MQ 


25ul of ligation mixture was used for transforming 100ul compentent cells. Good amount of colonies. Colonies were picked, grown overnight, and minipreped using the High Pure Plasmid Isolation kit from Roche.


Long weekend including Paris workshop.


David


Growing streptomyces for purification of the chaplin proteins.


Protocol: GrowingStreptomycesGR


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