Team:Groningen/1 June 2010

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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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