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Team:Groningen/1 June 2010
From 2010.igem.org
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| - | The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs. | + | The biobrick prefix and suffix are introduced into the pNZ8901 expression vector behind the spaS promoter using PCR and primers with 5’ overhangs. In this way, any biobrick with a RBS followed by a coding sequence can be brought to expression upon subtilin induction. |
Revision as of 21:59, 7 October 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 behind the spaS promoter using PCR and primers with 5’ overhangs. In this way, any biobrick with a RBS followed by a coding sequence can be brought to expression upon subtilin induction.
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
