Marie Curie Workshop Laboratory Manual Clostridial Gene ...

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Thursday 14 th SeptemberTODAYPerform the entire Day 7 protocol for Clostridium difficile, Clostridium sporogenesand Clostridium acetobutylicum.Day 7 – Induce target gene knockoutInoculate fresh selective broth with transconjugants and transformantsInoculate 1ml broths of appropriate anaerobic liquid growth media (supplementedwith 7.5µg/ml thiamphenicol to select for the re-targeted pMTL007 plasmid) with thetransformants and transconjugants as shown:Strain Inoculum MediumClostridium sporogenes10696 containingpMTL007::Csp-spo0A-249sClostridium acetobutylicumATCC824 containingpMTL007::Cac-spo0A-242aClostridium difficile630∆Erm containingpMTL007:: Cdi-spo0A-178a100µl overnight culture TYG + 7.5µg/mlthiamphenicol100µl overnight culture CGM + 7.5µg/mlthiamphenicol1 or 2 loops of cells BHI + 7.5µg/mlthiamphenicolNormally, these cultures are incubated at 37°C under anaerobic conditions until thereis visible growth, indicating the culture is in exponential phase (typically afterincubation for 1hr). However, to save time, we will omit this non-essential stepduring the workshop.Induce intron expression with IPTGAdd 10µl of 100mM IPTG stock to each culture, resulting in final IPTGconcentrations of 1mM. Incubate the cultures in the anaerobic cabinet forapproximately 3hrs.During the IPTG induction step:11:00 – 11:30 Coffee break11:30 – 12:30 Seminar12:30 – 13:30 LunchUniversity of Nottingham, Sept ’06 Page 16
Wash cells and allow recovery periodPellet the cells by centrifugation at 8000rpm for 1 minute. Discard the supernatants,then wash the cells by re-suspending them in 0.5ml of sterile PBS. Centrifuge asbefore and discard the supernatants.Re-suspend each pellet in 1ml of an appropriate anaerobic liquid growth media,unsupplemented with antibiotics, and incubate in the anaerobic cabinet forapproximately 3hrs.During the recovery incubation step:14:30 – 15:30 Seminar15:30 – 16:00 Coffee breakPlate integrants onto selective growth medium and incubate overnightPlate the integration mixtures onto fresh plates containing an appropriate anaerobicsolid growth medium supplemented with 2.5µg/ml erythromycin to select for presenceof the spliced ErmRAM, which indicates intron integration. Spread three plates with100µl of neat, 100µl of 100-fold diluted and 100µl of 5-fold concentrated integrationmixture.To determine the frequency of the integration event, serially-dilute each integrationmixture to 10 -4 , 10 -5 , 10 -6 and 10 -7 in 1.5ml tubes. Pipette 3x20µl drops of each of the10 -4 , 10 -5 , 10 -6 and 10 -7 dilutions onto one quarter of an appropriate plateunsupplemented with antibiotics. Do not invert the plates.Strain Selective medium Non-selective mediumClostridium sporogenes 10696spo0A knock-outsClostridium acetobutylicum ATCC824spo0A knock-outsClostridium difficile 630∆Ermspo0A knock-outsTYG + 2.5µg/mlerythromycinCGM + 2.5µg/mlerythromycinBHI + 2.5µg/mlerythromycinTYGCGMBHIIncubate the plates in the anaerobic cabinet until the end of the workshop.University of Nottingham, Sept ’06 Page 17
Page 1 and 2: Marie Curie Workshop Laboratory Man
Page 3 and 4: FOREWORDIt is with great pleasure t
Page 5 and 6: ProgrammeMonday 11 th September - A
Page 7 and 8: Overview of practical sessionsDurin
Page 9 and 10: 3. Perform PCR using the following
Page 11 and 12: Ligation of the digested PCR produc
Page 13 and 14: Wednesday 13 th SeptemberTODAYPerfo
Page 15: The thiamphenicol will select for t
Page 19 and 20: 1. Label PCR tubesLabel 8 PCR tubes
Page 21 and 22: Inspect plates and interpret result
Page 23 and 24: PARTICIPANTSSURNAME FIRST NAME INST
Page 25 and 26: IntroductionThe Centre for Biomolec
Page 27 and 28: If you hear the fire/evacuation ala
Page 29 and 30: Good Laboratory PracticeWithin the
Page 31 and 32: Waste DisposalAll hazardous biologi
Page 33 and 34: Mixed biological and chemical waste

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