Download DNA isol

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
CELL DISRUPTION METHODS
Technique
Example
effect
Cell type
Enzyme based
Lysozyme
Produces holes
Bacteria
Solution
Hypertonic salt
Cause rupture
Generally calls without
thick walls e.g.
Cultured animal cells
Detergent
SDS
Solubilises the
lipid bylayer
bacteria
Sonication
Sound waves
Disrupts
membranes by
high frequency
sound
Bacteria cells
homogenisation
Mechanical
shearing
Disrupts
membranes by
physical force
Plant tissue
biopsies
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
(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
5. Alcohol ppt
6. Purification e.g. phenol chloroform
7. Alcohol precipitation
Practical tips
1. 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.
How would you (what method
do you recommend and
why)
1. Lyse Pseudomonas cells
and obtain
a. genomic DNA
b. Plasmid DNA
2. Yeast cells to get genomic
DNA
3. Plant cells for genomic
DNA
4. DNA from Liver tissue