Molecular Biology

Tutorial 1

Phenol-Chloroform DNA extraction Method

Safety Precautions

We will occasionally use hazardous chemicals such as :

• Ethidium bromide : Carcinogenic

• Ethanol :Flammable

• Phenol:corrosive

• Chloroform :not to be inhaled as it cause liver cirrhosis

Safety Precautions cont.

• We regularly use equipment such as centrifuges and vortexes that can be hazardous when used incorrectly – make sure you are using all equipment correctly and be aware of what is going on around you.

• please wear gloves and eye protection when instructed to do so and always wash your hands before leaving the lab.

Micropipette usage

• When moving the plunger note that there are two levels:

-The first level will dispense the volume indicated on the pipette display. -The 2nd level gives an extra volume of air to expel any remaining traces of liquid.

Right procedures for using micropipettes

1- Hold Pipette in one hand. With thumb and forefinger, turn the plunger

button (on the top of the instrument) to set the volume.

2- Attach a new disposable tip to the pipette shaft using gloves. Press

firmly to ensure a positive airtight seal.

3- Depress the plunger to the first level to expel any trapped air to be

replaced by liquid of interest.

4- Holding the Pipette vertically, immerse the disposable tip into the

sample liquid to 3 mm deep.

5- Allow the plunger button to return slowly to the up/normal position.

Never let it snap up!

6- Pause briefly to allow the full volume of sample to be withdrawn into

the top.

7- Withdraw the pipette top from the sample liquid. Should any liquid

remain on the outside of the disposable tip, wipe it carefully with a

Kim-wipe, taking care not to touch the tip orifice.

8- To dispense sample, touch the tip end against the side wall of the

receiving vessel and depress the plunger slowly to the first level. Wait

two seconds, then press the plunger to the second level, to expel any

residual liquid in the tip.

9- With the plunger fully depressed, move the Pipette away from the

vessel carefully.

10- Allow the plunger to return to the normal position.

11- Discard the tip by depressing the tip ejector button. A fresh tip should

be used for each sample to prevent sample carryover.

Right procedures for using micropipettes (Continued)

Micropipette tips

• Accuracy refers to how closely the measured value of a quantity

corresponds to its “true” value.

ie.. 100 µl pipette is said to be accurate when picked up solution is

close enough to 100 µl.

• Precision expresses the degree of reproducibility, or agreement

between repeated measurements.

ie.. 100 µl pipette is said to be precise/reproducible, when the volume

of the picked up solution is identical in all these trials/times.

Accuracy and Precision

Experimental Procedures for measuring accuracy and precision

1.Prepare a clean 10 ml measuring cylinder, place it on a balance and

set the tare to 0. The balance must have an accuracy of at least 0.01 g.

2. Use a 10 – 100 µl automatic pipette, transfer 100 µl into the measuring

cylinder and record the weight. Set tare to 0 and repeat this 2 more times,

always noting the actual weight of transferred liquid.

3. Repeat the same experiment (use the same cylinder), but now transfer

100 µl using the 100 – 1000 µl pipette. Again, note the weight of the three

100 µl portions you transferred.

4. Enter your weights in the table below. Determine mean for both pipettes.

5. Explain any differences you may notice in the accuracies of the two

pipettes.

Pipette Volume Weight Mean

10-100 µl

100 µl

100 µl

100 µl

100-1000 µl 100 µl

100 µl

100 µl

Eppendorf tubes PCR tubes

Sterility • Biological molecules can be damaged or

degraded by enzymes that are found on non-sterile surfaces. Consequently, all manipulations are done with sterile materials (e.g. sterile tips and tubes)

• clean your bench with the provided 70% ethanol at the beginning and end of every lab period

• keep containers closed, except when removing the contents (i.e. do not leave the tip box open);

• do not touch the materials with your hands or any other non-sterile surfaces.

The cell

• All living things are made of cells.

• The three main parts of the cell are the cell membrane, nucleus and cytoplasm.

• The cell membrane, surrounds and protects the cell while nucleus surrounds DNA. Cytoplasm lies between both the nucleus and the cell membrane.

• All organelles are embedded in the cytosplasm such as mitochondria and ER.

Nucleic acid chemistry

• Deoxyribonucleic acid or DNA is the molecule that controls everything that happens in the cell.

• DNA contains the genetic code or commands that direct the activities of cells and this in turn has its ultimate effect on the body.

• DNA is present in all living things from bacteria to animals.

• In animals, it is found in almost all cell types, except red blood cells.

• DNA found within a nucleus in the cells.

• In order to extract DNA its necessary to denature and break down both the plasma and the nuclear membranes to release DNA.

• (Protein enzyme = protease) or other denaturing agent can be used to break proteins.

Purpose of DNA Extraction

To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc

From where can we extract

DNA?

•Blood

•Semen

•Saliva

•Urine

•Hair

•Teeth

•Bone

•Tissue(mouth cheeks)

DNA isolation

DNA must be separated from proteins and cellular debris.

Main DNA isolation methods:

• Organic extraction (Phenol:Chloroform method)

• Salting out

• Selective DNA binding to a solid support

Basic Protocol for all DNA isolation methods • The nucleus is protected within a nuclear membrane which is also

surrounded by a cell membrane Lipid bilayer). Four steps are used to remove and purify the DNA from the rest of the cell.

1. Lysis: Lysis is the disription/break of cell membranes to dissociate

nucleic acids from associated proteins and lipids and this would allow the release of nucleic acids.

2. Removal of contaminants such as proteins, lipids &

macromolecules by the use of either enzymes or chemical methods.

3. Precipitation of nucleic acids. 4. Washing (removal of residual/traces contaminants). 5. Resuspension of nucleic acids.

Phenol chloroform DNA extraction protocol (Organic method)

1. Lysis: SDS is an anionic detergent that ;

A- Denatures protein structure. B- Solubilizes lipids. C- Surrounds both denatured and lipids into a micelle. This would ultimately disrupt/break/pull apart both cellular and nuclear membranes due to the amphibathic (having both hydrophilic and hydrophobic Regions) character of SDS. NB1: EDTA; a chelating agent; is used in this step to chelate Mg ions which are essential For the Stability of both membrane & Nuclease enzymes. Chelating Mg will facilitate membrane disruption and prevent nuclaese enzymes from degrading nucleic acids too.

NB2: Cell debris and partially digested organelles are easily pelleted by centrifugation

leaving the cell extract as a reasonably clear supernatant.

2-Removal of contaminants

• This a series of steps where DNA is separated from the rest of the cellular components

• In the first part phenol/chloroform (1:1) is used to remove some of the proteins associated with DNA

• Phenol : denatures proteins and dissolves denatured proteins.

• Chloroform: is also a protein denaturant and solubilizes lipids.

• DNA is a polar molecule because of the negatively charged phosphate backbone.

• This polarity makes it more soluble in the polar aqueous phase and to be insoluble in organic solvents.

• When phenol is mixed with the cell lysate, two phases form. DNA partitions to the (upper) aqueous phase, denatured proteins and lipids partition to the (lower) organic phase.

• The aqueous solution of nucleic acid can be removed with a pipette.

• In the second part salts are added to interrupt the hydrogen bonds between the water and DNA molecules.

2-Removal of contaminants (Continued)

DNA is then precipitated from the protein in a subsequent step with absolute ethanol or isopropanol @ - 20° C. This occurs in the presence of salts that interrupts the hydrogen bonds between the water and DNA molecules

• In the presence of cations, ethanol induces a structural change in

DNA molecules that causes them to aggregate and precipitate out of

solution. •The DNA is pelleted by spinning with a centrifuge and the supernatant removed.

3-DNA precipitation

The precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts and other water soluble impurities but does not resuspend the DNA.

4- Washing

The clean DNA is now resuspended in either pure water or TE buffer (Tris EDTA) to ensure stability and long term storage.

5-Resuspension

Summary

Summary

Measurement of purity and DNA concentration:

• DNA concentrations can be accurately measured by UV absorbance at 260 nm.

• The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA sample.

• UV absorbance can also be used to check the purity of a DNA preparation.

• With a pure sample of DNA the ratio of the absorbance at 260 nm and 280 nm ( A260 / A 280 ) is ≥ 1.8 .

• Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein or with phenol.

• Also a pure sample of DNA would have a ratio of the absorbance at 260 nm and 230 nm ( A260 / A 230 ) is ≥ 1.8.

• Ratios of less than 1.8 indicate that the preparation is contaminated, either with ethanolic compounds, carbohydrates, guanidine or with phenol.

• .

Checking the Quality of your DNA

• The product of your DNA extraction will be used in subsequent experiments

• Poor quality DNA will not perform well in PCR

• You will want to assess the quality of your DNA extraction using the following simple protocol:

• Mix 10 µL of DNA with 10 µL of loading buffer

• Load this mixture into a 1% agarose gel

• Analyze results

1 kbp and 100 bp

ladders Genomic DNA of 5

species of cereals

Expected Results under UV Lamp Below is an agarose gel that has 5 genomic DNA samples from various plants. Note that the DNA runs at a very high molecular weight and as a clear, thick band. This DNA was extracted in a research lab under optimal conditions

If properly done, genomic extraction should result in bright bands in

the very high base pair range of a gel electrophoresis.

Sizes of Genomic DNA for

various Species in kbp

E. Coli 4,640,000bp

Yeast 12,100,000bp

Fruit Fly 140,000,000bp

Human 3,000,000,000bp

Pea 4,800,000,000bp

Wheat 17,000,000,000bp

The genomic fragments run at ~12kbp because they are sheared during extraction

Analyzing DNA Samples