Team:Heidelberg/Templates/MM week7

From 2013.igem.org

2013-06-10

Gel electrophoresis of the pLF03 digestion. Left: NEB 2 log DNA ladder; middle: digestion with EcoRI, right: digestion with XhoI+NdeI

• need to verify plasmid identity: digest pLF03 with EcoRI (for linearization) and XhoI+NdeI (verify insert size); 388 ng (4 µl of miniPrep from 2013-06-04) each
• insert only 3.3 kb in length, expected >4 kb (AccA1: 590AS * 3 = 1770 bp, PccB: 530AS * 3 = 1590 bp, CatR: 219 AS = 657 bp; adds up to 4017 bp, not counting RBS and spacers between the individual genes)
• contacted Lei Fang (Pfeifer Lab), there are NdeI and EcoRI sites in catR (they were not indicated in his plasmid map)
• to verify presence of chloramphenicol acetyl transferase in insert: transform BAP1 with 97 ng pLF03 (1 µl of miniPrep from 2013-06-04), plate on Amp + IPTG
• evening: pick BAP1-pLF03 colonies, transfer to Cm + IPTG
• inoculate 4 and 5 ml of liquid culture (Amp) with BAP1-pLF03

2013-06-11

Gel electrophoresis of the PCR amplificate. Lane 1: NEB 2-log ladder; lane 2: PCR with Phusion Flash; lane 3: PCR with Taq at 43°C annealing temperature; lane 4: PCR with Taq at 41.5°C annealing temperature

• BAP1-pLF03 grew on Cm + IPTG
• try to optimize PCR:
• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Phusion Flash F548 polymerase (performed by Dominik):

• run PCR of 5 ng pLF03 (1 µl 1:20 dilution of miniPrep from 2013-06-04) with primers ygfG21C1 and ygfG21C2 using Taq polymerase (cheaper):

• prepare glycerol stock of BAP1-pLF03
• make miniPreps of BAP1-pLF03 -> 21 ng / µl and 27 ng / µl in 27.5 µl

• received just plated DH5α-pCP20 from Sourjik Lab, grow at 30°C

2013-06-12

• purify Phusion Flash PCR product with Qiagen PCR purification kit -> 120 ng / µl in 27.5 µl
• inoculate 2 x 5 ml LB + Amp with DH5α-pCP20, grow at 30°C

2013-06-13

• miniPreps of DH5α-pCP20 -> 7.8 ng / µl and 14.6 ng / µl in 27.5 µl, respectively
• low yield of miniPreps: inoculate 2 x 5 ml LB + Amp with DH5α-pCP20, grow at 30°C

2013-06-14

• miniPreps of DH5α-pCP20 -> 25 ng / µl and 29 ng / µl in 27.5 µl, respectively
• prepare glycerol stocks of DH5α-pCP20
• to avoid electroporation with intact pLF03: add 10 µl of PCR amplificate (1.2 mg) on gel, perform gel extraction (QiaGen gel extraction kit) -> 3.2 ng / µl
• add 240 ng (2 µl) raw PCR product to 100 µl electrocompetent BAP1-pKD46
• add 16 ng (5 µl) gel-extracted PCR product to 100 µl electrocompetent BAP1-pKD46
• electroporate 50 µl each, add to 1 ml SOC with 1 mM IPTG
• shake at 300 rpm, 37°C for 1 h
• spin cells down (3 min 10 000 rpm = 8000 g), decant supernatant (leave about 200 µl supernatant in eppendorf tube)
• resuspend pellet, plate 100 µl on Cm-IPTG, leave rest at RT
• grow at 37°C

2013-06-15

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lane 2: negative control (BAP1-pLF03); rest: colony-PCRs from PCR-purified plate.

• one colony on plate with gel-extracted PCR product
• many colonies on plate with PCR-purified PCR product
• pick colonies from plate with PCR-purified PCR product, run colony-PCR (Taq, 25 µl total volume) with primers IK01 and IK02 (expected product: 465 bp):

Colony-PCR of BAP1-pKD46 electroporated with PCR amplificate of pLF03 grown on Cm+IPTG. Lane 1: NEB 2-log; lane 2: negative control (BAP1-pLF03); lane 3: colony-PCR of colony electroporated with gel-extracted PCR product; rest: colony-PCRs of colonies electroporated with PCR-purified PCR product.

• no product -> pick colony from gel-extracted plate, pick colonies from PCR-purified plate, run colony-PCR (Taq, 25 µl total volume) with primers IK01 and IK02:

• no product -> 2 possibilities:
  1. all colonies bear complete pLF03 plasmid (PCR was run with 5 ng template, DNA concentration in PCR product: 120 ng / µl, electroporated with 2 µl -> 1/6 ng pL03 vs. 240 ng amplificate)
  2. PCR did not work
• for colony from gel-extracted plate + 3 colonies from PCR-purified plate: add up to 3 ml medium, add IPTG (1 mM) + Cm, grow at 37°C
• plate rest of bacteria electroporated with gel-extracted PCR product on Cm + IPTG

2013-06-16

miniPrep of BAP1-pKD46 electroporated with PCR product from pLF03. Lane 1: NEB 2-log; lane 2: miniPrep of colony electroporated with gel-extracted product; rest: miniPreps of colonies electroporated with PCR-purified product.

• no colonies on gel-extracted plate
• make miniPreps of ON cultures, run gel
• pLF03: 9.2 kb, bands above 10 kb, bands smear, low intensity (applied all 30 µl of miniPrep to gel) -> fragments of genomic DNA?
• => PCR went wrong?