Team:Groningen/1 June 2010

From 2010.igem.org

(Difference between revisions)
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We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine
We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine
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<br>
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<pre>Week 22
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'''Week 22'''
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The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs. For optimization taq polymerase (fermentas) is used first
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The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs.
-
<br>
+
 
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<pre>Component µl Final concentration
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For optimization taq polymerase (fermentas) is used first
 +
Component µl Final concentration
Primer pNZ89bbs-for1 5 300nM
Primer pNZ89bbs-for1 5 300nM
Primer pNZ89bbs-rev1 5 300nM
Primer pNZ89bbs-rev1 5 300nM
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Template 0.5 ~14ng
Template 0.5 ~14ng
Taq 0.5 2.5u
Taq 0.5 2.5u
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MQ 29 </pre>
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MQ 29
- 94°C, 3’
- 94°C, 3’
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- 72°C,  10’
- 72°C,  10’
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[[Image:03-06-10 gn.jpg|200px|thumb|left|alt text]]
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No product. Likely the elongation step was too short for taq.
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<br>
+
 
 +
Repeat of previous PCR using the following cycling conditions
 +
 
 +
- 94°C, 3’
 +
- 94°C,  30’’
 +
30X - 50°C,  30’’
 +
- 72°C,  3’
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- 72°C,  10’
 +
 
 +
Gel 02-06-10
 +
Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use.
 +
Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid</pre>

Revision as of 21:40, 7 October 2010

iGEM Groningen 2010

Hydrophobofilm
pushing coatings into a greener future

We've started working on the wiki, been preparing plasmids and are settling in our office space. Note to self: should have brought coffee machine

Week 22

The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs.

For optimization taq polymerase (fermentas) is used first
Component	µl	Final concentration
Primer pNZ89bbs-for1	5	300nM
Primer pNZ89bbs-rev1	5	300nM
10x Taq buffer(-MgCl2)	5	1x
dNTP’s	2	200µM
MgCl2	3	1.5mM
Template	0.5	~14ng
Taq	0.5	2.5u
MQ	29	

- 94°C,	 3’
	- 94°C,  30’’
30X	- 50°C,  30’’
	- 72°C,  1’30’’
- 72°C,  10’

No product. Likely the elongation step was too short for taq.

Repeat of previous PCR using the following cycling conditions

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

Gel 02-06-10

Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use.
Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid










Week 23











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