Team:Heidelberg/Templates/MM week10

From 2013.igem.org

2013-07-01

• finish purification (digest for 5h, dissolve in 50 µl H₂O) -> 1162 ng/µl (performed by Fanny)
• prepare electrocompetent cells from ON culture (add 0.5% arabinose at dilution) (performed by Fanny)
• electroporate fresh electrocompetent cells, 1 aliquot from 2013-06-26 (23 µl DNA each) (performed by Fanny)
• grow in SOC at 30°C for 30 min, transfer to falcons, add 1 ml LB, add IPTG (1 mM), grow at 37°C for 4h
• spin cell down (8000 rcf for 3 min), decant supernatant, resuspend pellet in remaining medium
• plate on Cm+IPTG (2 plates for each sample: 10µl + rest, plates marked F: from -80°C, marked H: newly prepared cells), grow at 37°C

2013-07-02

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lanes 2, 3: negative control (BAP1-pLF03); lanes 4-9: colony-PCR of F-rest (lanes 8,9: from lawn); lanes 10-13: colony-PCR of H-10µl; lanes 2,4,6,8,10,12: primers IK01+IK02; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control)

• bacteria lawn on rest-plate from -80°C, possible colonies on other plates (barely visible, may be bubbles)
• pick 2 colonies from F-rest, sample of lawn, 2 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control) (20µl total volume):

• very evening: colonies on F-10µl, H-rest, H-10µl plates appear -> leave at 37°C ON

2013-07-03

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-4: negative control (BAP1-pLF03); lanes 5-13: colony-PCR F-10µl
bottom:lanes 2-4: colony-PCR of F-10µl; lanes 5-13: colony-PCR of H-10µl; lanes 2,5,11: primers IK01+IK02; lanes 3,6,12: primers IK01+IK03 (positive control); lanes 4,7,13: primers IK05+IK06 (negative control)

• colonies on F-rest plate appeared (plate left ON at RT)
• pick 4 colonies from F-10µl, 3 colonies from H-10µl, colony-PCR with primers IK01+IK02, IK01+IK03 (positive control), IK05+IK06 (negative control):

• F-10µl 1,2,4 show no negative control -> grow at 37°C
• continue growing plates at 37°C until evening, leave at RT ON

2013-07-04

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Lane 1: NEB 2-log
Top: lanes 2-10: cultures from 2013-07-03; lanes 2,5,8: primers IK01+IK02; lanes 3,6,9: primers IK01+IK03; lanes 4,7,10: primers IK05+IK06; lane 11: BAP1-pLF03 (negative control); lanes 12-13: colonies from F-10µl plate; lanes 11-13: primers IK05+IK06
bottom: lanes 2-13: primers IK05+IK06; lanes 2,3: colonies from F-10µl plate; lanes 4-6: colonies from F-rest plate; lanes 7-10: colonies from H-10µl plate; lanes 11-13: colonies from H-rest plate

• repeat colony-PCR for 3 ambiguous cultures form 2013-07-03 (use 1 µl of culture), pick 14 new colonies (only primers IK05+IK06); 20 µl total volume:

• no positives

2013-07-05

Restriction digest of pKD46. Lane 1: NEB 2-log; lane 2: EcoRI; lane 3: BamHI+NotI; lane 4: PstI+NotI; lane 5: undigested (225 ng (5 ml) loaded)

• digest 450 ng pKD46 (10 µl of 45.5 and 46 ng/µl miniPreps from 2013-06-06) with EcoRI (expected: 4816+1505 bp), BamHI+NotI (expected: 3675+2646 bp), PstI+NotI (expected: 3711+2363+243 bp); 30 µl total volume
• right plasmid
• inoculate 2 x 7 ml LB+Cm+IPTG(1mM) with colonies from F-10µl plate, grow at 30°C

2013-07-06

Restriction digest of BAP1-pKD46 electroporated with pLF03-PCR grown in Cm+IPTG(1mM), miniprepped.
Lane 1: NEB 2-log; lanes 2-5: Konrad's PCR; lane 6: digested with EcoRI; lane 8: undigested

• add Cm (1:3000), IPTG (1:1000) to liquid cultures, grow for 8h at 30°C
• make miniPreps -> 25 ng/µl and 14.4 ng/µl in 27.5 µl
• digest 25 ng/µl miniPrep with EcoRI (use all of miniPrep)
• load digest completely, 10 µl of 14.4 ng/µl miniPrep on gel
• no bands -> nanoDrop crappy, no plasmid
• wrong strain? Streak BAP1 on Cm, grow at 37°C