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Team:Groningen/1 June 2010
From 2010.igem.org
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, Arend Jan
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.
PCR:
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’
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.
PCR:
Component µl Final concentration Primer pNZ89bbs-for1 5 300nM Primer pNZ89bbs-rev1 5 300nM 10x pfu buffer(-MgSO4) 5 1x dNTP’s 2 200µM MgCl2 3 1.5mM Template 0.5 ~14ng Pfu polymerase 1 2.5u MQ 28.5 - 94°C, 3’ - 94°C, 30’’ 30X - 50°C, 30’’ - 72°C, 1’30’’ - 72°C, 10’
Good bands
