Team:Heidelberg/Templates/MM week18p

From 2013.igem.org

Contents

2013-08-26

Lane 1: NEB 2-Log, Lane 2: pIK6.1 digested with HindIII+BamHI; lane 3: pIK6.2 digested with HindIII+BamHI
  • make miniPreps of colonies 1 and 2: 71.4 ng/µl and 75.1 ng/µl in 38 µl
  • digest with HindIII+BamHI (3 µl DNA, 0.5 µl of each enzyme, 20 µl total); expected: 4.1 kb, 2.1 kb, 0.9 kb
  • no 0.9 kb fragment => no BamHI site in KanR gene, 2.1 fragment visible corresponds to acca-sfp => insert completely within backbone
  • co-transform TOP10 with pIK6.1+pRB21
  • sequences of pIK1.3 arrived: PIK1.3-2013-08-26.zip, PIK1.3-2013-08-26 VF2.clustal.txt, PIK1.3-2013-08-26 VR.clustal.txt
  • point mutation in permeability device, interestingly, both the frameshift in pIK1.2 and this point mutation create a stop codon at the beginning of the permeability device
  • send pIK1.4 to sequencing with primer VF2

2013-08-27

Lane 1: NEB 2-log, lane 2: pSB3K3-BBa_J04450 digested with EcoRI+SpeI
  • TOP10-pIK6.1-pRB21 are not blue, but the indigoidine group is having problems with pRB21 -> pRB21 might not work
  • sequence of pIK1.4 arrived: PIK1.4-2013-08-27.zip, PIK1.4-2013-08-27 VF2.clustal.txt
  • again frame-shift in permeability device -> send pIK1.6, pIK1.7, pIK1.10 to sequencing with primer VF2
  • make miniPrep of pSB3K3: 54.7 ng/µl in 38 µl
  • digest pSB3K3 with EcoRI+SpeI (20 µl total volume, 5 µl DNA, 0.5 µl of each enzyme)
  • gel-purify, ligate with pIK2.6 fragment from 2013-08-24 at RT for 1h -> pIK7:
what µl
pSB3K3 9
pIK2.6 8
T4 ligase 1 µl
T4 ligase buffer 2 µl
  • heat-inactivate: 75°C for 5 min
  • transform 10 µl of ligation into TOP10, plate on Kan, grow at 37°C

2013-08-28

Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 (-> pIK7) and primers VF2+IK25. Lane 1: NEB 2-log; lanes 2-6: pIK7
  • pick colonies, run colony-PCR with primers VF2+IK25 (expected: 1kb, iTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
35 95 30
54 30
72 90
1 72 600
1 10 inf
  • grow in 2xYT+Kan at 37°C

2013-08-29

Lane 1: NEB 2-Log, Lane 2: pIK7.1 digested with HindIII+BamHI; lane 3: pIK7.2 digested with HindIII+BamHI

2013-08-30

  • electroporate electrocompetent DH10ß with 1 µl, 14 µl of 1:4 Gibson mix for pIK1 from 2013-08-07
  • plate 10 µl, rest of each electroporation on Cm, grow at 37°C

2013-08-31

  • very large and small colonies present, after heat-schock transformation last time only large colonies present
  • pick 5 small colonies from each plate, grow in 2xYT+Cm (no 2xYT left => last 3 colonies from 14 µl / rest plate grown in LB+Cm) at 37°C

2013-09-01

  • pIK1.12 did not grow
  • make miniPreps of all cultures -> concentrations about 200-300 ng/µl in 37.5 µl (except pIK1.14: 50 ng/µl)
  • digest with BamHI+HindIII (20 µl total volume, 0.5 µl enzyme, 1 µl DNA)
  • expected fragments: 2151 bp + 7265 bp