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Miga moved page GMU:Intro to BioArt/Cloning to GMU:BioArt WS16/Cloning
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==Workshop 1 (Molecular cloning)== | ==Workshop 1 (Molecular/DNA cloning)== | ||
See also https://www.uni-weimar.de/kunst-und-gestaltung/wiki/GMU:BioArt/Sebastian_Kaye#DIY_PCR | |||
*1. cutting out DNA samples from different flesh sorts (sausage) | *1. cutting out DNA samples from different flesh sorts (sausage) | ||
*2. putting samples into Eppendorf reaction tubes (Epi) | *2. putting samples into Eppendorf reaction tubes (Epi) | ||
*3. adding buffer (1000 ul) enzyme (100 ul) in a bigger epi tube in order to prepare mix for dissolving samples | *3. adding buffer (1000 ul) enzyme (100 ul) in a bigger epi tube in order to prepare mix for dissolving samples (See 3-11: [[:File:DNeasy-IMG_3514.JPG]] for more info) | ||
*4. divide 200 ul of mix into tubes with samples so it dissolves all other proteins and leaves only dna | *4. divide 200 ul of mix into tubes with samples so it dissolves all other proteins and leaves only dna | ||
*5. putting samples into heating/cooling block for one hour with 56C | *5. putting samples into heating/cooling block for one hour with 56C | ||
*6. transfer liquid to the bigger epic tubes | *6. transfer liquid to the bigger epic tubes | ||
*7. adding 200 ul of | *7. adding 200 ul of solvent buffer to the dissolved samples. brakes the walls of the cell (functions as washing medium) | ||
*8. adding 200 ul 100% ethanol in order to get DNA out | *8. adding 200 ul 100% ethanol in order to get DNA out | ||
*9. transferring the liquid into the adapter tubes with powder (Ionenaustauschchromatographie) | *9. transferring the liquid into the adapter tubes with powder (Ionenaustauschchromatographie) | ||
*10. | *10. centrifuge/dremelFuge | ||
*11. 3 centrifuge cycles with different buffer; the last one with water in order to have dna in the final sample | *11. 3 centrifuge cycles with different buffer; the last one with water in order to have dna in the final sample | ||
*12. polymerase master mixer (half of the | *12. polymerase master mixer (half of the 2x master mix, 12,5ul + 12,5ul distilled water) + sample (10ul) + primer forward (2,5ul) + primer reverse (2,5ul). https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492 | ||
*PCR multiplication HOWTO: | |||
a) We start at 95C two strands divides, hydrogen bonds will break at this temperature; DNA is accessible | |||
<br>b) Annealing: primers bind to the targets Roughly at the 65C | |||
<br>c) 72C polymerase start elongation of dna. Copies into one direction. It is called TAQ polymerase; comes from specific species called /thermus aquaticus/; lives in gazers and very stable; For every 1000 base pairs you need to copy we need around 60s. | |||
<gallery> | <gallery> | ||
File:pcr-IMG_4694.JPG | File:pcr-IMG_4694.JPG | ||
File:pcr-IMG_4695.JPG | File:pcr-IMG_4695.JPG | ||
File: | File:LocustPcr_wo_quarz_Steckplatine.png | ||
File:pcr-IMG_4702.JPG | File:pcr-IMG_4702.JPG | ||
File:pcr-IMG_4706.JPG | File:pcr-IMG_4706.JPG | ||
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File:pcr-IMG_4713.JPG | File:pcr-IMG_4713.JPG | ||
File:pcr-IMG_4716.JPG | File:pcr-IMG_4716.JPG | ||
File:pcr-IMG_4728.JPG | File:pcr-IMG_4728.JPG | ||
File:0-kit.jpg | |||
</gallery> | </gallery> | ||
==Workshop 2 (Gel electroforese chamber)== | ==Workshop 2 (Gel electroforese chamber)== | ||
*1. cutting out from plastic box | *1. cutting out from plastic box (Fig. 1.) | ||
*2. preparing screws | *2. preparing screws to be put into the chamber (Fig. 2.); screws will be connected later to the power supply blue (-12V) and yellow (+12V) cables | ||
*3. preparing 1% agarose (agarose, | *3. preparing 1% agarose (agarose, TAE, distilled water); for 100ml solution use 1g agarose, 2ml 50x TAE buffer and 98ml distilled water | ||
*4. pouring agarose solution into the chamber | *4. pouring agarose solution into the chamber and adding SERVA DNA Stain G (1,5ul for 100ml solution) and mixing around so it spreads. | ||
*5. cutting out endings of agarose for screws (screws get hot when letting electricity through and therefore it needs liquid outside) | *5. cutting out endings of agarose for screws (screws get hot when letting electricity through and therefore it needs liquid outside) | ||
*6. pouring the liquid (distiled water + the solution; no agarose) | *6. pouring the liquid (distiled water + the solution; no agarose) | ||
*7. pipetting dna ladder for measurements into the first gel pocket | *7. pipetting dna ladder for measurements into the first gel pocket. https://www.addgene.org/plasmid-protocols/gel-electrophoresis/ | ||
*8. pipetting samples with colouring (instead of colour one could use 40% glicerol so the samples sink into the gel pockets) into the remaining gel pockets | *8. pipetting samples with colouring (instead of colour one could use 40% glicerol so the samples sink into the gel pockets) into the remaining gel pockets | ||
*9. | *9. let 24V flow through the chamber for 40 min (power supply blue+yellow cables) | ||
<gallery> | <gallery> | ||
File:electrophoresis-IMG_4736.JPG | File:pcr-IMG_4700.JPG|Fig. 1 | ||
File:electrophoresis-IMG_4736.JPG|Fig. 2 | |||
File:electrophoresis-IMG_4741.JPG | File:electrophoresis-IMG_4741.JPG | ||
File:electrophoresis-IMG_4751.JPG | File:electrophoresis-IMG_4751.JPG | ||
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</gallery> | </gallery> | ||
==Workshop 3 ( | ==Workshop 3 (Plant cloning)== | ||
man kann von einer Pflanze Millionen klonierten Pflanzen züchten. das wichtigste ist eine Zelle zu haben. | |||
*pflanzen Blätter ins Enyzm geben (Cellulase Macerozyme) | |||
*Enzyme kommen von microorganismen, sie teilen die Zellen (fressen Zellenwände) | |||
*Zellverband zerstören und auflösen damit wir einzelne Zellen naher haben | |||
*wenn das Milieu lebend/gut/günstig ist, können wir diese Zellen durch Teilung vermehren | |||
*am Anfang sieht man Callus (undifferenziertes pflanzen Gewebe) | |||
*man braucht bestimmte Pflanzenhormonmischung um daraus Mehrstoff | |||
*um ein Richtiges/siehtbares Pflanzen zu haben braucht man schon ein paar Monate. Und dafür sollte bestimmtes PH, Temperatur, Licht usw stehen | |||
*Wenn da zu viele Zellen sind, muss man erst zentrifugieren/waschen damit die Zellen vereinzelt werden könnten. Um das Milieu (=Umwelt / Nährlösung / Licht Temperatur / Belüftung) | |||
<gallery> | <gallery> | ||
File:plants-IMG_4416.JPG | File:plants-IMG_4416.JPG | ||