Transformation of 1 µl pPha-NR, 1 µl pPha-T1 and 1 µl pCAT (diluted 1:3 with H2O) in E. coli DH5α (30 min ice, 90 sec 42°C, 1 min ice, 45 min 37°C + 1 ml LB), plated on LBamp, oN 37°C.
2 colonies of pPha-NR and pPha-T1 inoculated in 5 ml LBamp, oN 37°C.
pCAT is a Cosmid, transformation failed.
Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 25 µl H2O.
Samples:
Digestion of pPha-NR with EcoRI and NcoI.
Digestion of pPha-T1 with EcoRI and NdeI.
The samples were incubated for 1 h at 37° C.
Gel substances
All positive.
Mutagenesis PCR of PfcpB (iBB3), PNR (iBB4), and TfcpA (iBB5).
Digestion of the template with 1 µl DppI.
Transformation of 1 µl PCR-product into E. coli DH5α (30 min ice, 60 sec 42°C, 1 min ice, 30 min 37°C + 1 ml LB), plated on LBamp, oN 37°C.
Transformation of iBB5 failed, new mutagenesis primers ordered.
3 colonies of iBB3 and iBB4 inoculated in 5 ml LBamp, oN 37°C.
Inoculation of Ca-competent E. coli DH5α in 2x5 ml LB, oN 37°C.
PPha-NR and pPha-T1 were inoculated in 5 ml LB, oN 37°C (for glycerol stocks).
Miniprep of of iBB3 and iBB4 (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl H2O.
3 ml E. coli DH5α pre-culture inoculated in 100 ml LB.
PCR on CAT-cassette (iBB2):
The relevant fragments were cut from the gel and then purified via “Qiagen Gel Extraction Kit” (Qiagen, Düsseldorf). Elution into 20 µl elution buffer.
Competent cells harvested and frozen.
Controlled with several Transformations:
30 min ice, 60 sec 42°C, 60 sec ice, 60 min 37°C + 1 ml LB; plated, oN 37°C.
Digestion of iBB3 with PvuII.
Digestion of IBB4 with PstI.
Expectations
IBB3 and iBB4 with point mutations prepared for sequencing:
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