Team:Heidelberg/Templates/MM week18p

From 2013.igem.org

2013-08-26

• 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, PIK1.3-2013-08-26 VR.clustal
• 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

• 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
• 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:

• heat-inactivate: 75°C for 5 min
• transform 10 µl of ligation into TOP10, plate on Kan, grow at 37°C

2013-08-28

• pick colonies, run colony-PCR with primers VF2+IK25 (expected: 1kb, iTaq, 20 µl total volume):

• grow in 2xYT+Kan at 37°C

2013-08-29

• sequences of pIK1 arrived: PIK1.6-pIK1.7-pIK1.10-2013-08-29.zip, PIK1.6-2013-08-29 VF2.clustal, PIK1.7-2013-08-29 VF2.clustal, PIK1.10-2013-08-29 VF2.clustal
• frame-shift in pIK1.6, point mutations in pIK1.7, pIK1.10
• perform miniPreps of pIK7 -> ca. 50 ng/µl in 38 µl
• digest 5 µl of pIK7.1, pIK7.2 with BamHI+HindIII (0.5 µl of each enzyme, 20 µl total volume)
• bands at right positions (expected: 4.1, 2.1, 0.8 kb)
• prepare glycerol stock of TOP10-pIK7.1

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