Team:Heidelberg/Templates/MM week11

From 2013.igem.org

Latest revision as of 02:22, 5 October 2013

2013-07-08

gel electrophoresis of PCR of pLF03 with primers IK07 and IK08 using Q5 polymerase. Product from one PCR tube was split to two neighboring lanes.

1 µl of gel-extracted PCR amplificate was loaded. Lane 1: NEB 2-log; lane 2: amplificate extracted by Dominik, lane 3: amplificate extracted by Ilia

• no colonies of BAP1 on Cm
• repeat PCR of pLF03:
• run 4 PCRs of 4 ng pLF03 (0.2 µl of 21 ng/µl miniPrep from 2013-06-11) with primers IK07 and IK08 using Q5 polymerase (50 µl total volume):

• extract amplificate from gel (4 lanes pooled, extracted by Dominik -> 119 ng/µl; 4 lanes pooled, extracted by Ilia -> 111.5 ng/µl)
• load 1 µl of each extract on gel

2013-07-09

• electroporate electrocompetent BAP1-pKD46 with 1µl, 5µl, 25µl DNA
• resuspend in 1 ml SOC + IPTG (1mM) + Ara (0.1%)
• grow for 90 min at 37°C, 400 rpm
• spin cells down, plate 10µl, rest of each sample on Cm+IPTG

2013-07-10

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lanes 2, 13: BAP1-pLF03 (control); lanes 3-12: colony-PCRs of large colonies from 25µl/10µl plate. Lanes 2-12: OneTaq; lane 13: iTaq

• on all plates: large + small colonies
• small colonies too small to pick => pick 10 large colonies from 25µl DNA / 10 µl bacteria plate, run colony-PCR with primers IK05+IK06 (Taq, 20 µl total volume):

• no integration
• continue growing plates at 37°C

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG.
Top: lane 1: NEB 2-log; lanes 2,4,6,8,10,12: primers IK05+IK06; lanes 3,5,7,9,11,13: primers IK01+IK03 (positive control); lanes 2,3: BAP1-pLF03 (control); lanes 4-7: colonies from 1µl/10µl plate; lanes 8-11: 1µl/rest; lanes 12-13: 5µl/10µl
Bottom: lane 11: NEB 2-log; lanes 1,3,5,7,9: primers IK05+IK06; lanes 2,4,6,8,10: primers IK01+IK03 (positive control); lanes 1-2: 5µl/10µl; lanes 3-6: 5µl/rest; lanes 7-8: 25µl/10µl; lanes 9-10: 25µl/rest

• when small colonies large enough: pick, run colony-PCR (OneTaq, 20µl total volume):

• 1st colony from 5µl/rest plate (marked 7) seems promising => grow at 37°C in 1 ml LB

2013-07-11

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB at 37°C. Lanes 1,4: primers IK01+IK03 (positive control); lanes 2,5: primers IK01+IK02; lanes 3,6: primers IK05+IK06; lanes 1-3: BAP1-pLF03 (control); lanes 4-6: colony 7; lane 7: NEB 2-log

• run full colony-PCR of liquid culture (OneTaq, 20 µl total volume, use 1 µl of culture):

• no integration
• 3rd population of colonies appeared on plates (stored at RT): very small, probably satellites

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG and then in LB at 37°C with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: large colony picked from 25µl/10µl plate; lane 4: colony marked 1 from 1µl/10µl plate; lane 5: colony marked 6 from 5µl/10µl plate; lane 6: colony marked 7 from 5µl/rest plate.

• run colony-PCR of liquid with primers IK07+IK08 (test for integration in wrong place) (OneTaq, 20µl total volume, use 1 µl liquid culture):

• no integration at all

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK05+IK06.
Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-9: colonies from 1µl/10µl plate; lanes 10-13: colonies from 1µl/rest plate
Bottom: lane 1: NEB 2-log; lanes 2-10: colonies from 1µl/rest plate (lane 5: swiped 3rd population of extremely small colonies)

• pick 20 new colonies, run colony-PCR with primers IK05+IK06 (iTaq, 20 µl total volume):

• unspecific bands (also present in colony-PCR from 2013-07-10)
• unspecific bands not present in BAP1-pLF03 PCR (control) and bottom lane 5 (satellite colonies) => not due to iTaq
• assume: some amount of satellites was picked together with colonies from plates -> give band at 500 bp; partially amplified ygfG fragments prime integrated insert
• do multiple sequence alignment of ygfG, [http://www.ncbi.nlm.nih.gov/nuccore/AF113605 pccB], [http://www.ncbi.nlm.nih.gov/nuccore/AF113603 accA1], and [http://www.ncbi.nlm.nih.gov/nuccore/AY048742 catR] using ClustalO => long enough stretches of partially identical sequences present
• add 1mM IPTG to cultures with colonies from 1µl/10µl plate picked today (grew in 500 µl LB at 37°C for several hours), after 2h at 37°C: streak on Cm+IPTG
• pick 10 colonies each from 1µl/10µl and 1µl/rest plates, transfer to new Cm+IPTG plate, grow at 37°C
• pick 1 colony from 1µl/10µl plate, inoculate 1 ml LB + Cm + IPTG (1mM)

2013-07-12

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG with primers IK01+IK02.
Top: lane 1: NEB 2-log; lane 2: BAP1-pLF03(control); lanes 3-12: colonies liquid culture that were plated on Cm+IPTG; lane 13: colony from 1µl/10µl plate that was transferred to new Cm+IPTG plate
Bottom: lane 1: NEB 2-log; lanes 2-6: colonies from 1µl/10µl plate that were transferred to new Cm+IPTG plate; lanes 7-12: colonies from 1µl/rest plate that were transferred to new Cm+IPTG plate; lane 13: liquid culture (Cm+IPTG(1mM)) from 1µl/10µl plate

• colonies grew slowly
• run colony-PCR with primers IK01+IK02 (iTaq, 20 µl total volume):

• no bands

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG with primers IK05+IK06. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lanes 3-4: colony-PCRs (from colonies marked 1 and 2)

• run colony-PCR with primers IK05+IK06 of 2 colonies from liquid culture that were transferred to Cm + IPTG plate (iTaq, 20µl total volume):

• band at 1.2 kb is now specific -> WTF?!?

2013-07-13

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK05+IK06. Lane 1: NEB 2-log; lanes 2,4,6,8,10,12: BAP1-pLF03 (control); lanes 3,5,7,9,11,13: colony-PCR; lanes 2,3: annealing at 63°C; lanes 4,5: 64°C; lanes 6,7: 65°C; lanes 8,9: 66°C; lanes 10,11: 67°C; lanes 12,13: 68°C.

• need to determine whether 1.2 kb band is specific: run gradient PCR of colony 2 (iTaq, 20 µl total volume) with primers IK05+IK06:

• intensity of 1.2kb band decreases, intensity of 0.5 kb band (control) does not -> 1.2 kb band is unspecific product
• check for presence of genomically integrated insert: run colony-PCR of colony 2 with primers IK07+IK08 (iTaq, 20 µl total volume):

• transfer some of colony 2 on Amp (check for presence of pLF03 as a whole), grow at 37°C
• transfer some of colony 2 to liquid culture: LB + Cm + IPTG (1mM), grow at 37°C

2013-07-14

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) with primers IK07+IK08. Lane 1: NEB 2-log; lane 2: BAP1-pLF03 (control); lane 3: colony-PCR

• bacteria grew on Amp => 3 possibilities:
  • Amp plates not selective
  • bacteria have complete pLF03 plasmid
  • bacteria still have pKD46, although grown at 37°C
• plate BAP1, BAP1-pKD46 on Amp, grow at 37°C
• colony-PCR did not work for the control -> repeat with OneTaq (use 1 µl of liquid culture)
• no amplificate for colony: possibly, Taq has problems amplifying 4.8 kb from genomic DNA

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG, grown in liquid culture and then transferred to Cm+IPTG (marked 2) and grown in liquid culture with Cm+IPTG(1mM). Lane 3: NEB 2-log; lanes 1,4: BAP1-pLF03 (control); lanes 2,5: 1 µl of liquid culture; lanes 1,2: primers IK07+IK08; lanes 4,5: primers IK01+IK06

• run colony-PCR with primers IK01+IK06 (OneTaq, 20 µl total volume, use 1 µl of liquid culture):

• control shows bright band where expected, colony does not => something is integrated, unknown what