Team:Heidelberg/Templates/MM week15p

From 2013.igem.org

Contents

2013-08-05

Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • received constructs from Matthew Mattozzi, Boston: pET21c-pcc-acca-mcce in NEB Turbo (Ampr), pWH8: mcc-pcs-pcc-acc-mmce in NEB Turbo (Kanr), E. coli MM17: MG1655 lambda(DE3) d(lacY) d(E14) mcr-pcs-pcc-acc::KanR integrated at phage site HK022 (Kanr)
  • plates contaminated with Drosophila larvae
  • re-streak on fresh plates, run colony-PCR for f3, f6 from pET21c-pcc-acca-mcce (Q5, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 98 120
35 98 5
61 10
72 120
1 72 600
1 10 inf
  • no bands

2013-08-06

Lane 1: NEB 2-log; lane 2: f3; lane 3: f6
  • run colony-PCR from pET21c-pcc-acca-mcce grown ON (Q5, 20 µl total volume) for fragments f3, f6:
Cycles temperature [°C] Time [s]
1 98 60
35 98 5
61 10
72 120
1 72 600
1 10 inf
  • inoculate 5 ml 2xYT+Amp with colony from which colony-PCR was run, grow at 37°C
  • no fragment for f3, re-run PCR (1 µl of liquid culture, 20 µl total, Phusion Flash):
Lane 1: NEB 2-log; lane 2: f3; lane 3: from genomic integration; lane 4: 2 µl of pET21c-pcc-acc-mmce miniPrep.
Cycles temperature [°C] Time [s]
1 98 60
35 98 1
65 5
72 60
1 72 600
1 10 inf
  • make miniPrep of liquid culture
  • no PCR product, miniPrep runs above 10 kb supercoiled (whole plasmid is expected to have 9 kb) (miniPrep has ca. 10 ng/µl)
  • re-run PCR for f3 from miniPrep (Q5, 0.2 µl DNA, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 98 60
35 98 5
61 10
72 120
1 72 600
1 10 inf

2013-08-07

Lane 1: NEB 2-log; lane 2: f3; lane 3: BamHI digest of pET21c-pcc-acc-mcce; lane 4: 1 kb plus ladder.
  • need to verify plasmid identity: linearize with BamHI (unique restriction site in both pET21c... and pWH8, use 5 µl of miniPrep, 30 µl total volume)
  • linearized pET21c... plasmid runs at 9 kb -> right
  • PCR of f3 worked -> hypothesis: Gibson overhang against permeability device bound to E. coli genome in colony-PCRs, as permeability device is mutated version of FepA, which is present in E. coli
  • gel-purify all fragments
  • f1: 54.2 ng/µl, f2: 64.4 ng/µl, f3: 24.4 ng/µl, f4: 14 ng/µl, f5: 47.6 ng/µl, f6: 25.2 ng/µl; all in 30 µl
  • perform Gibson assembly:
    • pIK1: 0.8 µl f1, 1.6 µl f2, 5 µl f3, 2 µl f4, 0.4 µl H2O, 10 µl NEB Gibson MM
    • pIK2: 1 µl f5, 6 µl f6, 3 µl f4
    • incubate at 50°C for 1h
  • transform TOP10: 2 transformations per plasmid: 5 µl of Gibson assembly, 5 µl of 1:4 diluted Gibson assembly
    • grow in 2xYT at 37°C, plate on Cm, grow at 37°C

2013-08-08

  • pIK1: several colonies, some of them pink -> pick 10 white, grow in 2xYT+Cm at 37°C
  • pIK2: 2 populations of colonies: large and small, several large colonies are pink -> pick 5 white large, 5 white small colonies, grow in 3 ml 2xYT+Cm at 37°C

2013-08-09

Lane 1: NEB 2-log; lanes 2-10: pIK1 miniPreps digested with BamHI+HindIII; lanes 11-13: large colonies of pIK2 miniPreps digested with BamHI+HindIII
Lane 1: NEB 2-log; lanes 2-3: large colonies of pIK2 miniPreps digested with BamHI+HindIII; lanes 4-8: small colonies of pIK2 miniPreps digested with BamHI+HindIII; lanes 9-13: Flo's PCR
  • colony 8 of pIK1 did not grow
  • make miniPreps of 2 ml everything else (2 ml each) -> ca. 62 - 600 ng/µl
  • miniPrep yields quite high, but consistent: pACYC with p15A origin (4 kb) [http://www.abgent.com.cn/downloads/BG0003-Plasmid-mini-kit.pdf yields 0.6 µg DNA / ml culture] in LB, culture in 2xYT/TB yields 2-5 times more cells (according to QiaGen) => ca. 10-15 µg expected for 9 kb plasmid
  • digest with BamHI+HindIII (20 µl total volume, 0.5 µl enzyme, 1 µl DNA [except for sample with 62 ng/µl, there 2 µl DNA was used])
  • expected fragments: 2151 bp + 7265 bp for pIK1, 2151 bp + 5029 bp for pIK2
  • send pIK1.2 and pIK2.2 to sequencing with primers VF2 and VR
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