Team:Heidelberg/Templates/MM week11

From 2013.igem.org

2013-07-08

• 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

• 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

• 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

• 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

• 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

• 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

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

• no bands

• 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

• 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

• 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

• 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