dna isolation. introduction dna isolation is an extraction process of dna from various sources. the...
TRANSCRIPT
DNA ISOLATION
INTRODUCTION
DNA isolation is an extraction process of DNA from various sources. The scientist must be able to separate the DNA from cell gently enough so that the DNA is not broken up or shredded.
DNA ISOLATION
DNA isolation is a procedure to collect DNA for subsequent molecular or forensic analysis. There are three basic steps in a DNA isolation.
BASIC STEPS IN DNA ISOLATION
DNA ISOLATION
STEP 1:
Breaking the cells open to expose the DNA within, such as by grinding the sample.
DNA ISOLATION
STEP 2: Removing membrane lipids by adding
a detergent.
STEP 3: Precipitating the DNA with an alcohol
— usually ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together.
DNA ISOLATION METHODS
Methods used to isolate DNA are dependent on the source, age, and size of the sample.
It can be isolated from any living or dead organism.
Common sources for DNA isolation include whole blood, hair, bones, nails, tissues, blood stains, saliva etc.
DOUBLE STRANDED DNA ISOLATION
DOUBLE STRANDED DNA ISOLATION
STEP 1: Pick a colony of bacteria harboring
the plasmid DNA of interest into a 12 X 75 mm Falcon tube containing 3 ml of media supplemented with the appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37C for 8-10 hours with shaking at 250 rpm.
DOUBLE STRANDED DNA ISOLATION
STEP 2:
Transfer the culture to an Ehrlenmeyer flask containing 50 ml of similar media, and incubate further for 11-14 hours.
DOUBLE STRANDED DNA ISOLATION
STEP 3:
Harvest the cells by centrifugation at 3000 rpm for 5 minutes in 50 ml conical tubes in the Beckman GPR tabletop centrifuge and decant the supernatant. The cell pellets can be frozen at -70degC at this point.
DOUBLE STRANDED DNA ISOLATION
STEP 4:
Resuspend the cell pellets in 2 ml of GET/Lysozyme solution, add 4 ml of alkaline lysis solution, gently mix, and incubate for 5 minutes in an ice-water bath.
DOUBLE STRANDED DNA ISOLATION
STEP 4:
Add 4 ml of 3M NaOAc, pH 4.8, gently mix by swirling, and incubate in an ice-water bath for 30-60 minutes.
DOUBLE STRANDED DNA ISOLATION
STEP 5:
Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal DNA by pouring through a double-layer of cheesecloth into a new 50 ml conical tube. Centrifuge at 3,000 rpm for 20 minutes at 4C in the Beckman GPR tabletop centrifuge.
DOUBLE STRANDED DNA ISOLATION
STEP 6:
Decant the supernatant to a 50 ml polypropylene centrifuge tube, add 20ml of a 20 mg/ml DNase-free RNase A and incubate in a 37C water bath for 30 minutes.
DOUBLE STRANDED DNA ISOLATION
STEP 7:
Add 7 ml (equal volume) of de-fined diatomaceous earth in guanidine-HCl (20 mg/ml) and allow the DNA to bind at room temperature for 5 minutes with occasional mixing. Centrifuge at 3,000 for 5 minutes in the Beckman GPR tabletop centrifuge.
DOUBLE STRANDED DNA ISOLATION
STEP 8:
8. Decant the supernatant, resuspend in 7 ml of diatomaceous earth-wash buffer, and centrifuge as above.
DOUBLE STRANDED DNA ISOLATION
STEP 9:
Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as above.
STEP 10:
Decant the supernatant and dry in a vacuum oven.
DOUBLE STRANDED DNA ISOLATION
STEP 11:
Resuspend the pellet in 0.6 ml of 10:1 TE buffer, and elute the bound DNA by incubation at 65degC for 10 minutes with intermittent mixing.
DOUBLE STRANDED DNA ISOLATION
STEP 12:
Remove the diatomaceous earth by centrifugation at 3,000 rpm for 5 minutes in the in the Beckman GPR tabletop centrifuge.
DOUBLE STRANDED DNA ISOLATION
STEP 13:
Transfer the supernatant to a 1.5 ml microcentrifuge tube and centrifuge at 12,000 rpm for 5 minutes in a microcentrifuge at room temperature. Transfer the supernatant to a new 1.5 ml microcentrifuge tube and ethanol precipitate.
DOUBLE STRANDED DNA ISOLATION
STEP 14:
Resuspend the dried DNA pellet in buffer and assay for concentration by agarose gel electrophoresis.
USES OF DNA ISOLATION
USES OF DNA ISOLATION
In Molecular Techniques:
Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes.
USAGE OF DNA ISOLATION
In Medicine:
Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes.
In medicine the latter application is the most common.
USAGE OF DNA ISOLATION
In Genetic Analysis:
On the other hand, forensic science needs to recover DNA for identification of individuals (for example thieves, accident, or war victims etc), paternity determination, and plant or animal identification.
THANK YOU