Crispr bacterial guide CIntroduction Genetic engineering involves modifying an organism ? s DNA to deliberately change an aspect of the organism for a particular purpose This kit demonstrates the power of the CRISPR Cas system by modifying the genomic DNA
CIntroduction Genetic engineering involves modifying an organism ? s DNA to deliberately change an aspect of the organism for a particular purpose This kit demonstrates the power of the CRISPR Cas system by modifying the genomic DNA of a strain of E coli so that it can grow and survive in conditions it normally would not be able to This kit requires hours of work over the course of at least days It can be completed in a weekend if fresh bacterial cultures are prepared on a Friday night As this document is constantly being updated with tips and pointers and there are video links embedded you can ?nd the most up to date version online at http goo gl YBfYuQ What is CRISPR Cas doing in this experiment Bacteria and all organisms need to make proteins to survive Proteins are tiny nanomachines that do everything from control our metabolism to keeping our heart beating In order to make a protein a cell uses the DNA code Each letters of DNA codes for a single amino acid and proteins are just chains of amino acids Proteins like cas are made by a nucleic acid and protein complex in the cell called the ribosome The media that you are attempting to grow the bacteria on contains a molecule called streptomycin which binds the ribosome and prevents it from making proteins not allowing the bacteria to replicate and so they can ? t grow This kit makes a speci ?c mutation in the ribosomal subunit protein rpsL that prevents streptomycin from binding it and so the bacteria can grow just ?ne on the media The genome of the E coli bacteria is that you will engineer is over million DNA bases in size and CRISPR will ?nd the single one that needs to be mutated This mutation will cause an amino acid change in the ribosomal subunit proteins that are being made For more information on sequences and details check out our more advanced CRISPR guide here CKit contents pg Timeline pg Making Plates pg Making Competent Bacteria pg DNA Transformation CRISPR pg Successful experiment example pg C LB Agar g in a mL tube LB Strep Kan Agar g in a mL tube mL glass bottle for pouring plates ?ll with mL water uL variable volume adjustable pipette uL increments Box count uL Pipette Tips Petri Plates Microcentrifuge tube rack Inoculation Loops Plate spreaders Pairs Nitrile Gloves in plastic bag microcentrifuge tubes mL microfuge tubes containing g LB mL centrifuge tube mL bacterial transformation bu ?er mM CaCl PEG DMSO Perishables E coli HME strain Cas and tracrRNA plasmid uL of ng uL crRNA plasmid uL of ng uL Template DNA uL of ng uL CPreparation hour Make plates set aside more time if it's your ?rst time making plates streak out bacteria onto an LB Agar plate takes min hours Let the bacteria grow easiest to just let it sit overnight Day of experiment Mix together sample plasmids and transformation
Documents similaires
-
25
-
0
-
0
Licence et utilisation
Gratuit pour un usage personnel Aucune attribution requise- Détails
- Publié le Oct 12, 2021
- Catégorie Law / Droit
- Langue French
- Taille du fichier 44.8kB