Team:Goettingen/week21-2

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#2 Speed Improvement - 21st week

V09_17_1 Preparative double digestion of flhDC, FliC, 18K-RFP and pSB1C3 followed by ligation

Experiment:
In order to clone the flhDC, FliC and 18K-RFP constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.

V09_17_2 Preparation of over night cultures

V09_18_1 Motility Assays

Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
Observation and Results:
Text

V09_18_2 Preparative double digestion of MotA, MotB, yhjH, 18C-RFP, 20E-RFP and 20I-RFP followed by ligation

V09_18_3 Chemical transformation of different plasmid-gene constructs into E. coli (DH10B) or (MG1655)

V09_19_1 Motility Assays

Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
Observation and Results:
Text

V09_19_2 Chemical transformation

V09_19_3 Preparation of over night cultures

Experiment:
Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
- pSB1C3-flhDC
- pSB1C3-fliC
- pSB1C3-18K-RFP

V09_20_1 Mini Prep and test digestion of pSB1C3-gene constructs

Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.

- pSB1C3-flhDC
- pSB1C3-fliC
- pSB1C3-18K-RFP
Observation and Results:
All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_20_2 PCR of fliC

Experiment:
In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

V09_20_3 Repetition of preparative double digestion of flhDC, FliC and pSB1C3

Experiment:
In order to clone the flhDC, FliC constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol.

V09_20_4 Preparation of over night cultures

V09_21_1 Ligation and chemical Transformation of flhDC, FliC and pSB1C3

Experiment:
The restriction products were separated on a gel in order to verify the digestions success and purify the desired fragments. After ligation of the genes into pSB1C3, we transformed the ligated plasmids into E. coli DH10B according to the protocol.

V09_21_2 Mini Prep and test digestion of pSB1C3-gene constructs

Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.All constructs were digested with XbaI and SpeI conducted as described in the protocol.

- pSB1C3-18C-motA (DH10B)
- pSB1C3-18C-motB (DH10B)
- pSB1C3-18C-yhjH (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20E-motA (DH10B)
- pSB1C3-20I-motA (DH10B)
- pSB1C3-20I-motB (DH10B)
- pSB1C3-20I-yhjH (DH10B)
- pSB1C3-RFP (BL21)
Observation and Results:
All three the pSB1C3-18K-RFP clones host the correctly inserted gene in the plasmid. The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.

V09_21_3 TITEL