bmt lab manual final

8/12/2019 Bmt Lab Manual Final

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EXP. NO. 1ISOLATION OF pUC 19 PLASMID FROM E. coli

DATE:

AIM:

To isolate the pUC 19 plasmid from E.coli culture cultivated in Luria Bertani broth.

REQUIREMENTS:

a) APPARATUS:Micro pipette, Eppendorf tubes, centrifuge, tips, Eppendorf stand.

b) CHEMICALS:Solution I, Solution II, Solution III, RNase, TE Buffer, Absolute alcohol, 70%

ethanol

PRINCIPLE:

Lyse the cells with sodium hydroxide (NaOH) and Sodium dodecyl sulphate (SDS).

SDS breaks the cell membrane. NaOH loosens the cell walls and releases the plasmid DNA

and sheared circular DNA. NaOH denatures the DNA. Circular DNA becomes linearized and

the strands are separated at alkaline pH 10-12. Plasmid DNA is circular and remains

topologically constrained. Sodium acetate or potassium acetate (KOAc), allows the circular

DNA to renature. Sheared circular DNA remains denatured as single stranded DNA (ss DNA).

The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt. Addingsodium acetate or potassium acetate to the SDS forms KDS, which is insoluble. This will

allow for easy removal of SDS from plasmid DNA. Ethanol or Isopropanol and a salt (such as

Ammonium Acetate, Lithium Chloride, Sodium Chloride or Sodium Acetate) allow DNA to

precipitate. DNA is negatively charged so adding a salt masks the charges and this will place

the DNA in the pellet.

The amount and concentration of isolated DNA can be determined spectrophotometrically.

DNA absorbs UV light so efficiently that optical absorbance can be used as an accurate and

rapid measurement of its concentration. A very useful approximation of absorbance is thatDNA at 50g/ml in aqueous solution has an A260 of 1.

Absorbance is also useful as a measure of purity of the DNA. The relevant spectrum isbetween 230nm and 320nm, having the absorbance at the maximum. If the absorbance

at 320nm is higher than the absorbance at 260nm presence of undesirable

contamination is indicated. The ratio of A260/A280 of a pure double stranded DNA

preparation should be between 1.8 and 2.0. Higher values are often due to RNA

contamination and lower values due to protein and phenol contamination. Pure

preparations of DNA have A260/A280ratio of 1.8 - 2.0. The concentration of the isolated

DNA is calculated according to the formula :


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Q (g/ml) = A260 x 50 x dilution factorMaterials and Solutions:-

LURIA BERTANI BROTH (LB)

Bacto- tryptone 10 g/L NaCl 10 g/L yeast extract 5 g /L Make the pH to 7.0 and autoclave For solid LB agar medium add 15 g/L agar before autoclaving.

SOLUTION I

50 mM GLUCOSE(Glucose should be added to maintain osmolarity and prevent the buffer

from bursting the cells. It increases the viscosity before lysing the cells, when viscosity

increases the probability of damaging DNA by mechanical forces decreases).

25 mM Tris-HClat pH 8.0 (Tris buffer is typical buffering substance for DNA having its

buffering capacity in the slightly alkaline range in which DNA can also be stored best

protecting from DNases (pH 8.0 - 8.2)).

10 mM EDTA(A chelator of divalent cations and thus DNases and RNases, which need

divalent cations for their function. It also functions in lysis of bacteria).

SOLUTION II

1% SDS(It is a detergent which denatures proteins and cell membranes).

0.2 N NaOH(raises the pH to about 12 so that the genomic DNA denatures but plasmid DNA

is still circular at this pH).

SOLUTION III

3.0 mM sodium acetate at pH 5.2 (neutralizing agent).

10 mg/ml RNase:

Dissolve 10mg of RNase A in 900 l of 10 mM sodium acetate (pH 5.0) and boil for 15 mins,

cool it to room temperature, add 100 l of 1M Tris-HCl (pH 7.5) and make it upto 1ml

volume. RNase at this concentration precipitates if boiled at neutral ph.

TE Buffer (pH 8.0)

10mM Tris-HCl, 1mM EDTA (pH 8.0), autoclave and store at RT.

Phenol: Chloroform: Isoamylalchol: : 25 : 24 : 1


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Denatures proteins from the mixtures

Absolute ethanol

Precipitates macro-molecules including DNA in presence of salts.

70% ethanol

Wash away the salts.

PROCEDURE:

1. Transfer 1.5 ml of culture into 1.5 ml eppendorf tubes.2. Centrifuge at 10,000 rpm for 1 min and carefully decant the medium.3. Add 100 l of solution I and resuspend by vortexing.4.

Add 200 l of solution II and mix completely by inversion. The cells should lyse andturn clear and viscous.

5. Let it stand for 3 mins on ice (but no longer, or the DNA will be irreversibly damagedand denatured) and then add 150 l of solution. Mix again by inversion till white clot

of DNA / Proteins / SDS complex forms.

6. Incubate on ice for 10 mins7. Centrifuge at 12,000 rpm for 10 mins8. Transfer the supernatant into a fresh 1.5 ml tube.9. To the solution add equal volume of phenol: chloroform: isoamyl alcohol (25:24:1).10.Mix gently and centrifuge at 12,000 rpm for 10 mins.11.Take the supernatant into a fresh tube and add 750 l isopropanol, mix well and

keep at -20C for 30 mins.

12.Resuspended the pellet in 50 l TE.

RESULT:


4/15

Exp.No-2AGAROSE GEL ELECTROPHORESIS

Date:

AIM:

To visualize the plasmid pUC 19 using agarose gel electrophoresis.

PRINCIPLE:

Agarose gel electrophoresis is used to separate and analyze the DNA. Electrophoresis is a

method of separating substances based on the rate of movement while under the influence

of electric field. Agarose is a polysaccharide purified from seaweed. The DNA to be analyzed

is forced through the pores of gel by the electric current. Under an electrical field, DNA will

move to positive electrode (red) and away from negative electrode (black) since DNA

naturally posses negative charge due to its phosphate backbone.

The DNA is visualized in the gel by addition of ethidium bromide (fluorescence dye). This

binds strongly to DNA by intercalating between the bases and it absorbs invisible UV light

and transmits the energy as visible orange light. A band is visible if it contains about 20ng of

DNA. The fluorescence yield is increased 20 to 30 fold when EtBr (Ethidium Bromide) binds

to DNA than free EtBr.

MATERIALS AND SOLUTIONS:

LOADING DYE:

Typical recipe for 5x dye.

25 mg bromophenol blue or xylene cyanol (Impart colour to sample). 4 g sucrose (increase density) Water to 10 ml.

Store at 4C to avoid mould growing on sucrose.

(The dye is negatively charged in neutral buffer and thus move in same direction as the DNAduring electrophoresis. Bromophenol blue migrates at a rate equivalent to 400 bp DNA on

0.8 % gel. If we want to see the fragments anywhere near this size (i.e., anything smaller

than 600 bp) then we use another dye because Bromophenol blue will obscure the visibility

of small fragment).


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ELECTROPHORESIS TANK BUFFER (TBE or TAE):

50 X TAE Buffer (1 L)

242 g Tris-HCl , 57.1 ml glacial acetic acid, 100ml 0.5 M EDTA (pH 8.0) , make the volume to

1l with water, autoclave and store at RT. Use 0.5 or 1x dilution for making and running theagarose gel.

10x TBE BUFFER (1L)

108 g Tris-HCl, 55 g Boric acid, 20ml of 0.5 m EDTA (pH 8.0), make volume to 1L with water,

autoclave and store at RT. Use 0.5X for agarose gel and 1X acrylamide gels. Discard the

stock solution if precipitation as crystals is seen during storage.

ETHIDIUM BROMIDE

10 mg/ml stock. Store in colored bottle and refrigerate.

PROCEDURE:

PREPARATION OF GEL:

1. Prepare agarose solution by weighing 1g agarose and dissolve in 100ml 0.5x TBE.2. Leave it to cool on the bench for 5 mins down to about 50C.3. Add 2 l of ethidium bromide (10 l/ml) and swirl to mix (The reason for allowing the

agarose to cool a little before this step is to minimize production of ethidium

bromide vapor).

4. Clean the tray, seal the tray using tap at both ends place the comb. Pour the gelslowly into the tank. Push any bubbles away to the side using disposable tip. Leave it

in RT for 30mins. Then remove comb and peel off tape.

RUNNING THE GEL:

1. Add an appropriate amount of loading buffer into each tube to a finalconcentration of 1X.

2. Load the sample with an appropriate marker. Close the gel tank, switch on thepower source and run the gel at 5V per cm.

3. Run the gel until the dye front reach 2/3rd of gel length. Visualize the DNA in UVtrans illuminator.

RESULT:


6/15

Exp. No-3RESTRICTION DIGESTION

Date:

AIM:

To digest the plasmid pCAMBIA 2300 and analyze the digested sample in agarose

gel.

PRINCIPLE

Restriction enzymes are the enzymes which have the ability to cut the specific

sequence on the DNA. Restriction endonucleases catalyze the cutting of the DNA molecule

by producing the symmetric breaking of the phospho-di-ester bonds of the both DNA strand

at the specific recognition sequence. Some Restriction endonuclease can generate the ends

that are staggered, with 5 or 3 protruding terminal nucleotides, other blunt, with full flush-

end nucleotides. Enzyme activity differs with temperature (25 to 37 oC). One unit is the

amount of enzyme required to digest 1g of DNA. Enzymes require cofactor for their

activity.

REQUIREMENTS:

a) APPARATUS:Eppendorf tubes, micropipettes.

b) CHEMICALS/REAGENTSWater, Buffer, Plasmid pCAMBIA 2300, endonucleases (HindIII, Eco RI)

Single Digestion (HIND III) Single digestion (EcoR I) Double digestion

Hin d III - 1 l Plasmid - 3 l Buffer - 2 l Water - 14 l

Eco R I - 1 l Plasmid - 3 l Buffer - 2 l Water - 14 l

Hin d III - 1 l EcoR I - 1 l Plasmid - 2 l Buffer - 2 l Water - 14 l

Note:To be added in the manner: Water,Buffer,Plasmid,Enzymes

Assay Buffer:

EcoRI- 50mM Tris-HCL, 100mM NaCl, 10 mM MgCl2, 5mM mercaptoethaol

HindIII10mM Tris-HCL, 50mM NaCl, 10mM MgCl2, 1mM DTT


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Dilution Buffer:

10mM Tris-HCl, 100mM KCl, 1mM EDTA, 1mM DTT, 50% glycerol, 200g/ml

BSA (ph. 7.4 at 25C)

PROCEDURE

1) Add DNA to the sterile water and mix three times.2) Keep it in ice.3) Prepare the reaction mix.4) Dispense the reaction mix to DNA, water aliquot uniformly.5) Vortex it mildly and spin briefly.6) Incubate the tubes at 37C for three hours.

RESULT :


8/15

Exp. No-4GENOMIC DNA EXTRACTION FROM PLANTS

Date:

AIM:

To isolate genomic DNA from neem leaves.

PRINCIPLE:

Isolation of plant genomic DNA is a requirement for most genome characterization

and mapping and isolation of genes for genetic engineering. The leaf tissues are first frozen

in liquid nitrogen and grinding with pestle and mortar helps to remove the cell wall and the

used of detergent like cetyltrimethyl ammonium bromide (CTAB) in the extraction buffer

disturbs all the cell membrane and releases the cell content. CTAB is a cationic detergent,

which aids in the lyses of cell membranes, and will form complexes with nucleic acids.

Sodium chloride aids in the formation of nucleic acid- CTAB complex. EDTA will chelate themagnesium ions, which is an essential co-factor for the enzymes and prevents the

indigenous endonucleases to act on nucleic acids. Mercaptoethanol is a reducing agent,

which protects DNA against quinines, disulphides, peroxidases, and polyphenol oxidases.

Poly vinyl pyrolidone (PVP) complexes with secondary plant products in particular complex

with polyphenols, tannins and quinines. The impurities such as RNA, proteins,

polysaccharides, pigments and tannins will be removed by treating with RNase, chloroform,

mercapto-ethanol and phenol. Selective precipitation of cell debris like pigments and

tannins is achieved by treating the extract with sodium acetate.

REQUIREMENTS:

a) APPARATUS:Micropipettes, eppendorf tubes, centrifuge, tips, eppendorf stand.

b) CHEMICALS/REAGENTS:1. CTAB extraction buffer (50 ml)

Tris-HCl (pH 8) 100 mM

Sodium Chloride 1.40 mM

EDTA (pH 8) 20 mMCTAB 2.0% W/V

2-mercapto-ethanol 0.1% W/V

(The extraction buffer must be autoclaved without 2-mercapto-ethanol, 2-mercapto-ethanol

should be added only prior to use)

2. TE Buffer

Tris-HCl (pH 8) 10 mM

EDTA (pH 8) 1 mM

3. Liquid Nitrogen


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4. RNase A solution 10 mg/ml5. Chloroform isoamyl alcohol 24:16. Ethanol 70%7. Absolute alcohol, isopropanol8. NaCl 3 M9. Sodium acetate (pH 5.2) 3 M

PROCEDURE:

1. Grind 1g of leaf tissue in liquid nitrogen using a mortar and pestle and transfer theground material to a 50 ml centrifuge tube. To the ground material, add 15 ml CTAB

extraction buffer, preheated to 90C and suspend thoroughly.

2. Incubate the suspension at 56C for 30 mins with occasional mixing. Cool the mixtureto room temperature add equal volume of chloroform: isoamylalcohol mixture (24:1)

and mix well to get an emulsion by inverting the tube several times for 15 mins.

3. Centrifuge the mixture at 10,000 rpm for 10mins and take out the aqueous layercarefully.

4. Precipitate the chromosomal DNA by adding 0.7 volume of isopropanol. Mix carefullyand keep at room temperature for 15mins. Retrieve the DNA with a glass hook or a

pipette. Transfer the DNA to a clean tube and air dry for 15 mins.

5. Dissolve the DNA in a minimal volume of TE buffer (up to 5ml depending upon theyield). Incubation at 65C will accelerate the dissolution of DNA and inactivate residual

DNase.

6. Add 1/100th volume of 10 mg/ml RNase A solution. Mix by inversion and incubate at37 C for 3 mins. Add equal amount of chloroform: isoamylalcohol (24:1), mix well and

centrifuge at 12,000 rpm for 10mins.

7. To the aqueous layer add (2 volumes of absolute alcohol and 1/10th volume 3Msodium acetate (if necessary).

8. Spin for 5mins at 10,000 rpm (if precipitation is not proper, the product can be kept infreezer overnight at -20C) Discard the supernatant. Wash the precipitate with 70%

ethanol.

9. Air dry the pellet and dissolve in TE buffer. Once resuspended in TE buffer the nucleicacid solution can be stored at 4C for upto 6 months or at -20 C for longer term

storage. Do not use -20C as standard storage conditions since continual freezing and

thawing leads to shearing of the DNA.

RESULT


10/15

Exp. No: 5ISOLATION OF GENOMIC DNA FROM HUMAN BLOOD

Date:

AIM:

To isolate genomic DNA from human blood sample.

PRINCIPLE:

DNA is extracted from white blood cells as RBCs lacks nucleus. Lysis buffer lyses the blood cells.

The role of the proteinase k is the primary concern here. As there are lot of contaminating

proteins present, these contaminants must be removed for the purity of the DNA as well as the

long term storage.

Proteinase (Broad spectrum of serine protease) digests the contaminating proteins. In addition,use of proteinase k degrades the nucleases and protects the nucleic acids fromattack.

REQUIREMENTS:

a) APPARATUS:Eppendorf tubes, micropipettes, centrifuge, vortex spin

b) CHEMICALS/REAGENTS1. RBC lysis buffer

15.5 mM ammonium chloride

10.0 mM potassium bicarbonate

1mM EDTA

2. Proteinase K Buffer pH 7.620mM Tris HCl

4mM Na2EDTA

10mM NaCl

3. 10%SDS4. Proteinase K 20mg/ml5. 5.3M NaCl

PROCEDURE:

1) 250 l of blood is added to eppendorf tube.2) To this 750 l of lysis buffer was added.3) After mixing, it was kept on ice for 15 min, occasionally inversion was done.4) Centrifugation was done at 4,500 rpm for 15 minutes.


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5) The supernatant was discarded and to the pellet, 750 l of the proteinase k buffer and50 l of SDS was added.

6) To this 5 l of proteinase k was added and kept in water bath at 60C for 15 min.7) 500 l of NaCl was added and then vortexing was done.8) Centrifugation was done at 4,500 rpm for 20 minutes.9) Supernatant was transferred to the fresh eppendrof tube.10)Equal volume of isopropanol was added.11)Inversion was done 5-6 times and was centrifuged at 4,500 rpm for 15 minutes.12)The supernatant was discarded and 70 % ethanol was added.13)Centrifugation was done at 4,000 rpm for 15 minutes.14)Supernatant was discarded and the pellet was dissolved in 200 l of distilled water.

RESULT


12/15

Exp. No-6SPECTROPHOTOMETRIC QUANTIFICATION OF DNA

Date:

AIM:

Quantitative and Qualitative estimation of DNA using spectrophotometer.

PRINCIPLE:

Nucleic acids (DNA and RNA) has maximum absorbance at 260 nm. One OD value (standard)

corresponds approximately 50 g/ml of double stranded DNA, 40g/ml of single stranded DNA/

RNA and 20 g/ml of oligonucleotides. The ratio between the readings at 260 nm and 280 nm (OD

260/OD 280) provides an estimate of the purity of nuclei acid. Pure preparations of DNA and RNA

have a ratio of approximately 1.8 and 2.0 respectively. If the DNA is contaminated with protein the

ratio will be2.0 indicates that the DNA is contaminated with RNA. The optical

density (OD).

REQUIREMENTS:

a) Apparatus:

Spectrophotometer, cuvette, eppendorf tubes, micropipettes, pipette tips.

b) Chemicals/reagents:

sterilized water, Plant and Human DNA samples.

PROCEDURE:

1. Take two eppendorf tubes and number them as 1 and 2.2. Add 100L of sterile distilled water to tube 1 and use it as blank.3. Take 98 L of sterile distilled water in eppendorf tube 2 and add 2 L of DNA.4. Mix well by pipetting.5. Switch on the photometer and choose the program ds DNA. 6. Set the dilution.7. Read the optical density/quantity of DNA (g/mL).8. Also note the OD values for the following A230, A260, and A320.


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CALCULATION;

Extinction coefficient for DNA is 50.

RESULT:

RESULT/INFERENCE:

FOR HUMAN GENOMIC DNA FOR PLANT GEOMIC DNA

A230

A260

A280

A320

A260/280

Concentration =g/ml

A230

A260

A280

A320

A260/280

Concentration = ..g/ml


14/15

Exp. No-7COMPETENT CELL PREPERATION

Date:

AIM:

To prepare E.coli Top 10 competent cells

PRINCIPLE:

Since E. coli is not naturally transformable, the ability to take up DNA or competency must be

induced by chemical methods using divalent and multivalent cations (calcium, magnesium,

manganese, rubidium, or hexamine cobalt). Alteration in the permeability of the membranes

allows DNA to cross the cell envelope of E. coliwhich is composed of an outer membrane, an

inner membrane, and a cell wall. The outer membrane of E. colican be understood by

application of the fluid mosaic model for membranes and is composed of phospholipids,

proteins, and lipopolysaccharides. Many channels or zones of adhesions are formed by the

fusion of the outer membrane and the inner membrane through the cell wall layer. These

channels allow for the transport of DNA molecules across the cell membrane. The negative

charges of the incoming DNA, however, are repelled by the negatively charged portions of the

macromolecules on the bacteriums outer surface. The addition of CaCl2serves to neutralize the

unfavorable interactions between the DNA and the polyanions of the outer layer. The DNA and

competent cells are further incubated on ice for thirty minutes to stabilize the lipid membrane

and allow for increased interactions between calcium ions and the negative components of the

cell. The reaction mixture is then exposed to a brief period of heat-shock at 42oC. The change in

temperature alters the fluidity of the semi-crystalline membrane state achieved at 0oC thus

allowing the DNA molecule to enter the cell through the zone of adhesion

REQUIREMENTS:

a) Apparatus:

eppendorf tubes, micropipettes, pipette tips, cooling centrifuge, water bath

b) Chemicals/reagents:

1. 100mM CaCl2

2. LB broth(Luria-Bertani broth)

per liter of water:

Bacto-tryptone 10 g

Bacto-yeast extract 5 g

NaCl 10 g


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Adjust pH to 7.5 with NaOH and autoclave for 20 minutes. LB plates, add 15 g

of agar to LB broth before autoclaving. For LB plates with antibiotic, add the appropriate amount

of the selective antibiotic to sterile LB-agar media that has been precooled to 50oC

PROCEDURE

1. Freshly streak E.colitop 10 cells on LB agar medium containing streptomycinantibiotic (50ug/ml)

2. Inoculate a single well isolated colony of E.colicells into 5ml LB broth withstreptomycin antibiotic 50ug/ml. This is the mother inoculum

3. Incubate the tube at 37C in a shaker over night4. 1/100thof the culture is inoculated into 50ml LB broth with streptomycin antibiotic

50ug/ml

5. Allow the cell to grow until OD600=0.6 is reached6. Transfer 3ml of culture to eppendorf tube and spin at 5000rpm for 10 minutes at 4C7. Discard the supernatant and to the pelleted cells add 500l of 100mM CaCl28. Keep the tubes on ice for 10 mins and spin again at 5000 rpm for 10 mins at 4C9. Discard the supernatant and to the pellet add 60l of CaCl2and 40l of 60% sterile

Glycerol

10.Store the samples at -70CTransformation

1. Add 1l of plasmid DNA (50ng) and incubate in ice for 30 minutes2. Keep the tubes in a water bath at 42C and heat shock for 90 seconds3. Snap cool the tubes in ice for 2 minutes4. Add 900l of autoclaved LB broth and incubate the tubes at 37C with shaking for 1

hour

5. Take 150l of culture and plate on LB agar medium containing ampicillin antibiotic(100ug/ml)

6. Then keep the plates overnight at 37C7. Look for colonies the next day

RESULT:

bmt lab manual final

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