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STEPS IN A MOLECULAR BIOLOGY PROJECT 1. Isolate DNA or RNA

STEPS IN A MOLECULAR BIOLOGY PROJECT 1. Isolate DNA or RNA extraction and isolation methods e.g. alkaline SDS, high salt,…. 2. Manipulate DNA cut and paste with enzymes clone using vectors amplify the piece of DNA of interest in vitro systems 3. Analyse DNA probe/blot sequence

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STEPS IN A MOLECULAR BIOLOGY PROJECT 1. Isolate DNA or RNA

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  1. STEPS IN A MOLECULAR BIOLOGY PROJECT • 1. Isolate DNA or RNA • extraction and isolation methods e.g. alkaline SDS, high salt,…. • 2. Manipulate DNA • cut and paste with enzymes • clone using vectors • amplify the piece of DNA of interest • in vitro systems • 3. Analyse DNA • probe/blot • sequence • sequence comparison • 4. Produce gene product of manipulated DNA • fermentation technology • protein analysis • 5. Use the information to create strategies for treatment e.g. gene therapy

  2. CELL DISRUPTION METHODS

  3. STEPS IN: (A) DNA EXTRACTION 1. Cellular lysis (e.g. detergent) 2. Chelating agents e.g. EDTA/citrate 3. Proteinase agents e.g. proteinase K 4. Purification of DNA e.g. phenol chloroform 5. Alcohol ppt (B) RNA EXTRACTION 1. treat reagents e.g. DEPC- diethyl pyrocarbonate 2. Cellular lysis (e.g. detergent) 3. Rnase treatment e.g. Rnasin / Vanadium compounds 4. RNA solvents e.g. Guanidinium salts 5. Proteinase agents and DNase agents 6. Purification e.g. phenol chloroform 7. Alcohol precipitation

  4. Practical tips • Know the experiment’s level of forgiveness. Another way of saying that it pays to know the chemistry of your procedure. Inevitably, each experiment has a degree of forgiveness, which is a really useful thing to know. This allows you to gauge your level of care, which in turn will reflect on your efficiency as well as your ability to troubleshoot. 2. Get your sample as pure as you can. Although this is related to point one, consider the following thought: any impurity is an outright invitation for caveats. i) If you have contamination, your subsequent steps may be hindered, or worse, unduly affected. ii) If you have a contamination, you might just lose your sample outright. i.e. DNA doesn’t like nucleases. And nucleases are“everywhere.” 3. If you had to choose, be gentle rather that be rough. Doesn’t hurt to be careful when handling material. i.e. keep everything cold, since these enzymes are much more active at physiological temperatures. (i.e. use of "ice cold" this and that") Wear gloves, etc. 4. Know the idiosyncrasies of your molecule: At times, you need to be aware of specific nuances that apply to your particular “brand” of molecule. For instances, genomic DNA is different from plasmid DNA is different from a PCR product 5. Think carefully about how much stuff you actually need. Small amounts generally easier to work with, machines are smaller, take less time to operate, etc, etc, etc… So, if you know, you don’t need much, then only use that amount.

  5. How would you (what method do you recommend and why) • Lyse Pseudomonas cells and obtain • genomic DNA • b. Plasmid DNA • 2. Yeast cells to get genomic DNA • 3. Plant cells for genomic DNA • 4. DNA from Liver tissue

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