Team:Heidelberg/Templates/MM week20p

From 2013.igem.org

2013-09-09

• co-transform TOP10 with pRB22.3 and pIK7.1, plate on Cm+Kan, grow at 37°C

2013-09-10

• after ca. 18h at 37°C: lots of tiny colonies, not blue
• leave at RT in drawer (no light)

2013-09-11

• colonies became blue

TOP10 cotransformed with pRB22.3 and pIK7.1

• start constructing pIK8: promoter even weaker than the one used by Cambridge 2007, weak RBS
• run PCRs of (2 ng DNA, 20 µl total volume, using Q5):
  • pIK2.6 with primers IK40+IK43 -> f7

• • BBa_I746200 with primers IK41+IK42 -> f8

2013-09-12

Lane 1: NEB 2-log; lane 2: f7; lane 3: f8

• run gel, gel-purify fragments -> 6.3 ng/µl in 20 µl (f7); 36.1 ng/µl in 20 µl (f8)
• perform Gibson assembly: 8 µl f7, 2 µl f8, 10 µl MM, incubate at 50°C for 1h
• dilute Gibson mix 1:4, electroporate electrocompetent DH10ß with 1 µl, 14 µl of dilution
• plate 10 µl, rest on Cm, grow at 37°C

2013-09-13

• very large and very small colonies present, pick starting with small colonies, run colony-PCR with primers IK26+VR (iTaq, 20 µl total volume) using 2 ng of BBa_I746200 in pSB1AK3 as positive control:

• clones grown in eppendorf tubes in 2xYT+Cm at 37°C

2013-09-14

Colony-PCR of DH10ß transformed with pIK8 and primers IK26+VR. Lane 1: NEB 2-log; lanes 2-11: colonies; lane 12: positive control (BBa_I746200 in pSB1AK3)

• run gel
• colonies designated pIK8.1, pIK8.2, pIK8.6, pIK8.9 positive
• transfer to baci falcons, add 2xYT+Cm, grow at 37°C

2013-09-15

Restriction digest of pIK8 with BamHI+HindIII. Lane 1: NEB 2-log; lane 2: pIK8.1; lane 3: pIK8.2; lane 4: pIK8.6; lane 5: pIK8.9

• make miniPreps: 73.5 ng/µl (pIK8.1), 12.9 ng/µl (pIK8.2), 289.0 ng/µl (pIK8.6), 169.0 ng/µl (pIK8.9) in 37.5 µl each
• digest with BamHI+HindIII using 2 µl (pIK8.1), 10 µl (pIK8.2), 1 µl (pIK8.6), 1 µl (pIK8.9) DNA (20 µl total volume)
• expected: 2.1 + 7.3 kb