Team:Heidelberg/Templates/MM week19p

2013-09-02

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: [http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_signalling#Plan they tried different promoter strengths], [http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Signalling_project_description#Standard_Construction_of_Permeability_Device 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

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