AMITY UNIVERSITY RAJASTHAN JAIPUR

AMITY INSTITUTE OF BIOTECHNOLOGY

BATCH 2009-2013

Molecular Biology Laboratory User Hand-Out

By Dr. Girish Gowswami

INDEX

EXPERIMENT 1
Prepare buffer for genomic DNA isolation from plant (TAB method) and bacterial culture (E. coli)

Objective:
To prepare buffer for genomic DNA isolation for plant (CTAB METHOD) and bacterial culture (E. coli)

Requirements:
Tris base , acetic acid, EDTA, KOH, Boric Acid, CH3CooNa, NaCl, CTAB, distilled water, glassware, etc.,

Theory:
A buffer solution is the one that resists a change in the pH on the addition of acid (H ) or base (OH ) more effectively than an equal volume of water. TAE (Tris-acetate-EDTA) buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA. IT is used in agarose electrophoresis typically for the separation of the nucleic acids such as DNA and RNA. TBE (Tris-Borate-EDTA) buffer is a buffer solution containing a mixture of Tris base, boric acid and EDTA. Borate in the TBE buffer is a strong inhibitor for many enzymes. TAE has lower buffer capacity than TBE and can easily become exhausted, but inner double stranded DNA runs faster in TAE. CTAB (hexadecyl trimethly ammonium bromide) is a detergent that helps lyse the cell membrane, however it is pretty poor with proteins denaturing, and so something else can be used.
+

Methodology:
For preparing 50ml of 50X TAE add: 1. Tris base: 12.1 g 2. Acetic acid: 2.855 ml 3. 0.5 EDTA ( Shake vigorously ): 5 ml 4. Adjust pH to 8.5 by KOH For preparing 50 ml of 5X TBE add: 1. Tris base: 2.65 g 2. Boric acid: 1.37 g 3. 0.5M EDTA: 1 ml 4. Adjust pH 8 For preparing 50 ml of CTAB buffer add: 1. CTAB:1 g

2. 1M Tris : 5 ml (pH 8) 3. 0.5M EDTA: 2 ml (pH 8) 4. 5M NaCl: 14 ml Make up the volume to 50 ml by adding distilled water

Precaution:
1. Never pipette any solution with mouth. 2. Before adjusting pH, check that pH meter is well calibrated properly. 3. Handle the glassware and chemicals carefully, especially EDTA.

EXPERIMENT 2
Agarose gel electrophoresis for quantitatively analysis at DNA.

Objective:
Agarose gel electrophoresis for quantitative analysis at DNA.

Requirements:
Agarose, TAE Buffer, EtBr, glassware, electrophoresis unit

Theory:
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate mixed population of the DNA and RNA fragments or to separate protein by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate further than longer ones because shorter molecules migrate more easily through pores at the gel. Proteins are separated by the charge because of the pores of the gel are too large to sieve the proteins.

Methodology:
For DNA isolation, 0.7% - 1% of Agarose is used 0.85 g Agarose + 50 ml (TAE, 1X) Heat the solution to melt the agarose Cool down to 55 o C 60 o C Add 2.5 l to ETBR Pour solution to tray with comb

 Let it solidify; put in buffer tank Load the DNA Sample (1l dye + 2 l DNA) Run between 60 V 100 V for 45 minutes to 1hour. See bands under UV- Light

Precautions:
1. Heat the solution carefully so that it wont spill out of the beaker. 2. Handle EtBr very carefully and always use gloves when handling EtBr since it is highly carcinogenic. 3. Comb must be placed carefully, so that proper wells can be obtained for sample loading.

Results:
The given DNA samples were electrophoresed and the distinct DNA bands were observed when seen under UV light.

EXPERIMENT 3
Isolate gnomic DNA from plant leaves.

Objectives:
To isolate genomic DNA from plant leaves.

Requirements:
Plant leaves, CTAB, Buffer, water bath, glassware, chloroform, iso amyl alcohol, centrifuge, ammonium acetate, absolute ethanol, DNAse, RNAse

Theory:
DNA extraction from plant tissue culture can vary depending on the materials required. The essential any mechanical means of breaking down the cell wall and membrane allows access to the nuclear material without its degradation for this, usually an initial grinding stage with liquid nitrogen is employed to break down cell wall material and allows access to the DNA which is harmful cellular enzyme and chemicals remain inactivated. 1. Grind 200mg of plant tissue to a fine paste in approximately 500 l of CTAB buffer. Transfer CTAB / plant extract mixture for about 15 minutes at 55 0 C in a recirculating water bath. 2. Incubate the CTAB / plant extract mixture for about 15 minutes at 55 0 C in a recirculating water bath. 3. After incubation, spin the CTAB /plant extract mixture at 12000 rpm for 5 min to spin sown cell debris. Transfer the supernatant to clean micro-fuge tube. To each tube add 250 l of chloroform: iso-amyl alcohol (24:1) and mix the solution by inversion. After mixing spin the tubes at 13000 rpm for 1 min. Transfer the upper aqueous phase (it contains DNA) to a clean micro-fuge tube. 4. To each tube add 50 l of 7.5M acetate followed by 500 l of ice cold ethanol. 5. Invert the tubes slowly several times to precipitate the DNA. Generally, the DNA can be seen to precipitate outside the solution. Alternatively, the tubes can be seen to be place for 1 hour at 200C after addition of ethanol to precipitate the DNA.

6. Following precipitation, the DNA can be pipette off by slowly rotating the tip in the cold solution. The precipitated DNA sticks to the pipette and can and is visible as a clear thick precipitate. Into a micro-fuge tube containing 500 l of ice cold 70% ethanol and slowly invert the tube. Repeat, alternatively, the precipitate can be isolated by spinning the tube at 13000 rpm for a minute to form a pellet/ Remove the supernatant and wash the DNA pellet by adding the 2 changes ice-cold 70% ethanol. 7. After the wash, spin the DNA into a pellet by centrifuging at 13000rpm for 1 min. 8. Remove all the supernatant and allow the DNA pellet to dry (Approx 15 min.) of the genomic DNA that has been isolated from plant leaves. 9. Re-suspend the DNA in the sterile DNAase free water (approx 50 l 400 l of water); the amount of water needed to dissolve the DNA can be very depending on how much is isolated. RNAase A ( 10 g/ ml) can be added to water prior to dissolve the DNA, to remove any RNA in the preparation (10 l/ RNAase in 10 ml of water). 10. After the resuspension the DNA is incubated at 65 0 C for 20 min, to destroy any DNAase that may be present and stored at 4 0C. 11. Agarose gel electrophoresis at the DNA will show the integrity of the DNA while spectrophotometery will give the indication of the concentration and cleanliness.

Result:
Distinct DNA bands were seen when the gel was viewed in the presence of UV light, of the genomic DNA that has been isolated from plant leaves.

Precaution:
1. Pipette out the supernatant carefully. 2. Prevent over-drying of the DNA. 3. Handle the chemicals carefully.

Experiment 4
To perform restriction digestion using isolated plant DNA sample

Objectives:
To perform restriction digestion using isolated plant DNA sample.

Requirements:
Restriction enzyme (EWRI), restriction buffer, double distilled water, water bath, glassware etc,.

Theory:
A DNA fragment resulting from cutting of a DNA strand by a restriction enzyme (restriction endonucleases ), a process called restriction digestion. Each restriction enzyme is highly specific, recognizing a particular short DNA equence or restriction site and cutting both the DNA strand at specific points within these site. Most restriction site are pallindromic ( the sequence of the nucleotide is the same on both strands when read in 5 to 3 direction) and are four to eight nucleotides long restriction fragments which can be analyzed using techniques such as gel electrophoresis or used in recombinant DNA technology.

G A A T T C C T T A A G

Methodology:

i.
2. 3. 4.

The components are mixed in the following order to get restriction digested enzyme.
EWRI buffer : 2 l DNA sample : 10 l EWRI enzyme : 1 l

1. Distilled water(sterilized) : 12 l

ii. Incubate in water bath maintained at 37 0 C for 1 hour. iii. Cast the gel and load the sample for electrophoresis at 60 V for 45 min to 1 hour. iv. See the gel under the UV light.

Result:
DNA bands can be seen under the UV light, of the restriction fragments obtained.

Precautions:
1. All the components to be added accurately. 2. Maintain water bath at 37 0 C or else the DNA would be denatured.

EXPERIMENT 5
To isolate the bacterialgenomic DNA.

Objectieves:
To isolate the bacterial genomic DNA.

Requirment:
Cell culture, SDS, proteinase K, T.E. Buffer, glassware,etc.,

Theory:
Bacterial Dna is double helical. Bacterial DNA is typically in the form of plasmids a circular form of double stranded DNA. The DNA is packaged in loops back and forth. The bundled DNA is called nucleoid. It concentrates the DNA in part of the cell, but it is not separated by a nuclear membrane. The DNA does from loops back and forth protein core, attached the cell wall. The DNA is accessible to enzymes that make RNA and proteins. In the bacterial cell, the DNA gets transcribed to protein before it is completed.

Methodology:
Take culture in 1ml distilled water in Eppedort. Centrifuge at 5000 rpm for 5 minutes. Take pellet cells.

 Resuspend pellet in 467l T.E. Buffer, 30l 10% SDS, 3l protienase k. Incubates for 1 hour at 370C Add equal volume of phenol/chloroform by inverting tube. Spin at 12000 rpm for 15 minutes. Transfer supernatent to Eppendrot. Add 1/10 volume of sodium accetate and mix by gentle immersion. Add equal volume of isopropanol. Incubate at 00C for 30 minutes or overnight. Discard supernatent and air dry. Dissolve pellet in 40l of T.E. Buffer. Perform Agarose Gel Electrophoresis.

Result:
Isolated bacterial DNA bands were seen when observed under UV light after running gel.

Precautions:
1. Pure cell culture to be taken. 2. Incubates for the given time only. 3. Decant supernatant carefully.

EXPERIMENT 6
Demonstration of the gel documentation system.

Objective:
Demonstration of the gel documentation.

Theory:
Gel doc is also known as gel documentation System or Gel Imager is widely used in molecular biology for the imaging and the documentation at nucleic acid and protein, agarose or polyacrlyamide gel typically stained with EtBr or fluorescents such as SyBr Green Generally, gel doc is with EtBr or light trans-illuminator, a hood to shield external light sources and a camera for images capturing.

Principles:
With fluorescent straining nucleic acids, a florescent substance that has bound to nucleic acids id excited by UV irradiation and emits florescent light. The florescent substance EtBr binds specifically to nucleic acids and the amount of bonding depends on the molecular weight and concentration of the nucleic acids. In other words, a band for a large molecular weight or large amount will shine brighter; conversely fluorescence will be weaker for a small molecular weight or small amount.

Disappearance of band due to UV

With continued irradiation at UV rays, the fluorescence of a band gradually weakens.. This is particularly striking when the molecular weight or the amount at samples is small. It even disappears in 20-30 in seconds to about 1 minute due to UV radiation at 25 mm. The freeze button provided can be minimize UV irradiation time, it provided. Purpose of Use: 1. Photography of stained gels. 2. Printout of photographic data. 3. Saving of photographic data. Image data is displayed in the real time. Some samples that can be filmed are: 1. Fluorescent stained samples requiring UV excitation. Ex; EtBr, SyBr Green. 2. Samples having bands stained by using a white trans-illuminated. Ex; CBB, Silver stain. 3. Samples having membrane or TLC are coloured using in-cabinet lamp. EX; Antigen-Antibody,

Application:
1. Rapid DNA, RNA, protein imaging 2. Easy multiplex western blot imaging. 3. State imaging. 4. Accurate automated imaging.