DNA isolation from plants and gel electrophoresis

DNA extraction is a procedure used to isolate large amounts of DNA from cell.

DNA can be isolated from plant and animal cells as well as from bacteria and other living beings

What we will be doing? Isolation of a good amount of DNA from plant cells.

DNA extraction

Plants contain large amount of secondary metabolites such s polyphenolic compunds and polyscaccarides.

It is more difficult to extract DNA from halophytes due to presence of huge amounts of salt in the cells.

 Plant cell walls can be very difficult to disrupt. It often contain significant amounts of compounds such as tannins, phenolics, and complex polysaccharides that can affect DNA quality and inhibit downstream reactions.

If not handled carefully, the DNA may be broken down or degraded during the extraction.

Isolation of plant DNA is difficult… why?

Protocol The most commonly used DNA extraction protocols involve breaking

(through grinding) or digesting away cell walls and membranes Removal of membranes lipids is facilitated by using detergents such as

sodium dodecyl sulphate (SDS), Cetyl trimethyl-ammonium bromide (CTAB) or mixed alkyl trimethyl-ammonium bromide (MTAB).

The released DNA should be protected from endogenous nucleases Getting rid of other compounds that interferes in the extraction of DNA

◦ Proteinase K addition and denaturation at 65C followed by precipitation by Phenol and chloroform.

◦ RNAs removal◦ Polysaccharide removal ◦ DNA can be separated from other compounds via centrifugation◦ Ppt formation◦ Analysis of DNA on agarose gel with a ladder

Spectrophotometer measures the intensity of absorbance of DNA solution at 260 nm wavelength, and also indicates the presence of protein contaminants but it does not tell whether the DNA is degraded or not.

Outcomes at the end of any DNA extraction There is no DNA. The DNA appears as sheared (too

fragmented), which is an indication of degradation for different reasons.

DNA appears as whitish thin threads (good quality DNA) or brownish thread (DNA in the presence of oxidation from contaminants such as phenolic compounds).

Liquid nitrogen Pestle and Mortar 2% CTAB ( Cetyl trimethylammonium bromide) Chloroform and Isoamylalchol (24:1) TE buffer Isopropanol Washing buffer Eppendorf tubes Falcon tubes

Protocol

Washing and grinding Weigh (1g) of leaves and wash them in distilled water. Let

them dry and then grind them in liquid nitrogen using pistill and mortar.

Procedure

CTAB incubation and centrifugation Incubate 2% CTAB (25ml) at 65C. Add plant material to it. Shake it in water bath at 62C for half hour. Allow mixture to cool for 3 minutes at room temperature. Washing Now add 25ml chloroform and isoamyl alcchol (24:1). Mix and centrifuge it at 400rpm for 20 minutes. Separate upper solution into a new falcon tube. Now add equal volume of chilled isopropanaol and mix it

slowly.

Keep it in room temperature over night or in refrigerator for half an hour.

Centrifuge the sample for 15 minutes at 4000 rpm. Now add 1 ml washing buffer to wash pellet after

discarding the supernatant. Keep it at room temperature for 20 minutes. Put mixture in an eppendorf. Now centrifuge (9000rpm for 5 minutes). Now incubate at 37C for 25 minutes untill pellet

become dry. Add 600-700 ul TE to dissolve the pellet.

Isolation

Ultra pure DNA Ready to use genomic DNA for enhanced performance in

sensitive applications Low amounts of (chemical) waste Less time consumption Easy to handle

Advantages of the protocol

Electrophoresis is a common lab technique, used for the separation and analysis of macromolecules like DNA, RNA and Protein.

DNA samples are placed in a special gel and subjected to an electric field. 

DNA is negatively-charged.

The shortest segments will travel farthest, while the longest fragments will remain closest to the origin.

Gel electrophoresis

Electrophoresis chamber, gel form and comb Power supply that produces 50-150 volts Agarose DNA sample Ethidium Bromide TAE buffer Tape Pipettes Tips Flask

Material and Chemicals

Add 1.2 gram agarose to 120ml TAE buffer. Heat the solution in the microwave oven on high power

until it comes to a boil. Place tape across the ends of the gel form and place the

comb in the form. Pour cooled agarose into the form. Add Ethidium Bromide into it.

The agarose should come at least half way up the comb teeth.

When the agarose has solidified, carefully remove the comb. Remove the tape from the ends of the gel form and place it in the chamber.

Load the sample in each well along with loading buffer. Allow the gel to run for 30 to 45 minutes. Finally, observe the gel and DNA bands.

Procedure

Avoid grinding violently otherwise DNA will be broken down. Time management is important. Do not harshly mix the extracted DNA in isopropanol in order

to have good quality DNA. Wash the pellet in wash buffer carefully. When removing melted agarose from oven keep in mind to

protect your hand with a pot holder or folded paper towel and gently swirl it to re-suspend any settled agar. 

Immediately rinse the agar flask. When the agar has solidified, carefully remove the comb.  Do not forget to remove the tape from the ends of the gel

form. Make a written record of which sample you will load in each

well of the gel.

Precuations