Team:Heidelberg/Templates/MM week19p

From 2013.igem.org

2013-09-02

• send pIK1.11, pIK1.13, pIK1.14 to sequencing with primer VF2

2013-09-03

• sequences arrived: PIK1.11-pIK1.13-pIK1.14-2013-09-03.zip, PIK1.11-2013-09-03 VF2.clustal.txt, PIK1.13-2013-09-03 VF2.clustal.txt, PIK1.14-2013-09-03 VF2.clustal.txt
• insertions leading to stop-codon in pIK1.11, pIK1.13; two bases of RBS missing in pIK1.14
• check wiki of Cambridge 2007 team, who constructed the permeability device: they tried different promoter strengths, only the weakest promoter gave colonies => bacteria might be selecting against the permeability device
• put pIK1.12 in incubator again, maybe it has the right sequence and grows slowly
• pIK1.12 did grow, make miniPrep -> 67 ng/µl in 38 µl
• send pIK1.14 to sequencing with primers IK25, VR

2013-09-04

Lane 1: NEB 2-log; lanes 2: pIK1.12 digested with BamHI+HindIII

• sequences arrived: PIK1.14-2013-09-04.zip, PIK1.14-2013-09-04 IK25.clustal.txt, PIK1.14-2013-09-04 VR.clustal.txt
• sequences match
• prepare glycerol stock of DH10ß-pIK1.14
• digest pIK1.12 with BamHI+HindIII (20 µl total volume, 134 ng DNA [2 µl of miniPrep from 2013-09-03], 0.5 µl enzyme)
• digest looks about right, send to sequencing with primer VF2

2013-09-05

• sequence arrived: PIK1.12-2013-09-05.zip, PIK1.12-2013-09-05 VF2.clustal.txt
• ca. 1 kb missing from end of permeability device