MOLECULAR BIOLOGY

INDEX
Serial No	Date	Name of The Experiment	Page No
01	28.06.2010	Estimation of protein concentration in supplied sample by lowry method	2-
02	29.06.10	Isolation of nucleic acid from E. coli cell and determination of concentration of isolated nucleic acid (DNA & RNA) by spectrophotometer.
03		Separation and visualization of DNA through agarose gel electrophoresis.

Experiment No.  01	Date: 28.06.2010
Name Of The Experiment: Estimation of protein concentration in supplied sample by Lowry method.

Principle:

    Lowry method is used for the determination of protein containing phenolic hydroxyl group. Determination of phenolic hydroxyl group total amount of protein is determined. This is relatively more effective method.

The reaction occur in two steps:

Step- 1:

    Reaction with copper in alkali

    Cu2++protein                   protein Cu2+ complex (Bluish yellow).

Step- 2:

    Reaction of copper treated protein with phosphomolybdate phosphotungstate reagent and form color complex. The intensity of color depends on the amount of these aromatic amino acids present in protein and will thus vary from different proteins.

    Protein + Cu2+ + folin-phenol reagent                  Reduction product                                     (blue)

    The reaction occurs at alkaline PH 6 formed color complex can be measured at wavelength 750 nm. Measuring the absorbance the total amount of the protein is determined.

    This method was discovered by Lowry in 1951.

Apparatus:

1. Beaker of 100ml or 50ml.
2. Volumetric flask of 100ml/50ml.
3. Test tube.
4. Pipette.
5. Measuring cylinder (100ml).
6. Spectrophotometer.

Reagents:

1. For reagent A: 2% Na2co3 in 0.1N NaOH solution.
2. For reagent B: 1% CuSO4.5H2O solution.
3. For reagent C: 1% Na-K tartrate solution.
4. For reagent D: Reagent A: Reagent B: Reagent C

=    100    :    1    :    1

5. For reagent E: Phenol reagent (2N): Water.

= 1:1 (5ml: 5ml).

6. A standard Bovine Serum albumin solution.

1. Preparation of reagent A:

    Reagent A is prepared by dissolving 2gm Na2co3 in 0.4gm 0.1N NaOH solution and the volume is made upto 100ml by adding distill water.

Calculation:

    10mg of Bovine Serum albumin (BSA) was dissolved into 50ml of distilled water.

So,

    50ml solution contains 10mg of protein.

    1ml        "        "  =          mg   "

    0.2ml        "      "  =                mg   "

                                  =     μg

    = 40 μg of Protein

    Similarly, 0.4ml, 0.8ml, 1.2ml, 1.6ml solution contain 80, 160, 240 and 320μg of protein.

For 0.4ml solution:

    0.2ml solution contain 80Mg of protein

1ml        "        "  =          μg   "

0.4ml        "        "  =               μg   "=    = 80 μg

For 0.8ml solution:

    0.2ml solution contain 80Mg of protein

1ml        "        "  =          μg   "

0.8ml        "        "  =               μg   "

       = 160 μg

For 1.2ml solution:

    0.2ml solution contain 80Mg of protein

1ml        "        "  =          μg   "

1.2ml        "        "  =               μg   "

       = 240 μg

For 1.6ml solution:

    0.2ml solution contain 80Mg of protein

1ml        "        "  =          μg   "

1.6ml        "        "  =               μg   "

       = 320 μg

2. Preparation of reagent B:

    Reagent B is prepared by dissolving 1gm CuSO4.5H2O in 100ml distilled water.

3. Preparation of reagent C:

    Reagent C is prepared by dissolving 1gm Na-K tartrate in some portion of distilled water and after dissolving the volume is made upto 100ml by adding sufficient distilled water.

4. Preparation of reagent D:  

    Reagent D is prepared by mixing 100ml of reagent A, 1ml of reagent B and 1ml of reagent C.

5. Preparation of reagent E:

    It is commercially available 2N solution. It can be prepared by diluting commercial 2N phenol reagent with equal amount of water (5ml phenol reagent + 5ml distilled water).

6. Preparation of stock solution:

    A standard Bovine Serum Albumin was prepared by dissolving 0.01 gm/10mg Bovine Serum Albumin in 50ml distilled water. So, the concentration of the stock solution is 200μg/ml or 0.2mg/ml.

Procedure:

1. Preparation of standard solution:

    Five test tubes were taken and marked as A, B, C, D, E and F. Then 0ml, 0.2ml, 0.8ml, 1.2ml and 1.6ml stock solution taken in test tube respectively and then adding 2ml, 1.8ml 1.6ml, 1.2ml 0.8ml, 0.4ml distilled water. Thus the concentrations of the solution are 20μg/ml, 40μg/ml, 80μg/ml, 120μg/ml and 160μg/ml.

    Then another six test tube were taken and marked as A, B, C, D, E and F and 1ml of standard solution from each of the test tube were taken respectively. Then 5ml of reagent D was taken in each of the six test tubes. Then vortex for 1 minute and kept it standing at room temperature for 10 minutes.

    Then 0.5ml of reagent E (Freshly prepared) was added to each of the test tube and then vortex 2 minutes and allow for standing at room temperature for 30 minutes.

2. Preparation of sample solution:

    1ml of sample solution was taken in a test tube from the unknown test tube. Then 0.5ml reagent D is added and vortex 1 minute and allow standing at room temperature for 30 minutes.

    Finally added 0.5ml of freshly prepared reagent E and vortex 2 minutes and allow standing at room temperature for 30 minutes.

3. Then the absorbance of blank, standard and sample solution were taken at 750 nm in the spectrophotometer. At 1st the absorbance of blank solution was taken. Then the readings of the absorbance of the stardard and sample solution were taken.

4. Curve:

    A standard curve was constructed by plotting the concentration versus absorbance. Then the amount of protein in the supplied sample was calculated from the curve.

5. Data:

Serial no.	Solution to be observed	Absorbance
1. 2. 3. 4. 5. 6. 7.	Blank 20 μg/ml 40 μg/ml 80 μg/ml 120 μg/ml 160 μg/ml Sample

Result:

    The concentration or amount of protein in supplied sample was  

       μg/ml

Experiment No.  02	Date: 29.06.2010
Name of the Experiment: Isolation of nucleic acid from E. coli cell and determination of concentration of isolated nucleic acid (DNA & RNA) by spectrophotometer.

Principle:

    A Nucleic Acid is a macromolecule composed of chains of monomeric nucleotides. These molecules carry genetic information or form structures within cells. The most common nucleic acids are DNA & RNA. Nucleic Acids are universal in living things as they are found in all cells and viruses. Friedrich Miescher first discovered Nucleic Acid.

    The extraction and purification of nucleic acids from biological material requires lysis of cell membrane in activation of the cellular nucleases and separation of the desired nucleic acids from cellular debris. Cellular lysis can be accomplished through enzymatic treatment or detergents.

    Traditionally, the subsequent extraction of the nucleic acids from the cellular lysate involves the use of phenol or phenol/chloroform (CHCl3). These solvents preferentially separate the nucleic acids into the aqueous phase from the other cellular components including proteins, found in the organic phase. The isolated nucleic acids (DNA, RNA) in the aqueous layer are concentrated by ethanol precipitation.

    As nucleic acids show absorbance at UV-range the concentration determination is done through spectrophotometer at 260 nm And it should be taken into consideration that 1 OD260= 50 μg/ml.

    The equation used for calculating concentration is as follows-

Concentration of nucleic acids = Absorbance × 50 × dilution factor

Limitation:

    This procedure does not work well with Gram Positive Cocci.

Reagents:

For preparation of Luria broth (L.B.):

1. Yeast extract
2. NaCl
3. Tryptophan
4. Distilled water

For preparation of E. coli solution:

1. Luria broth (L.B)
2. E. Coli colony

For isolation of nucleic acids:

1. 10% SDS (Sodium dodecyl sulphate)
2. 10N NaOH
3. TE (Tris Edetate)
4. Phenol: chloroform (1:1)
5. 5M NaCl
6. 95% ethanol
7. Distilled water
8. Overnight culture of E. coli.

Apparatus:

For preparation of Luria broth (L.B):

1. Bottle
2. Autoclave

For preparation of E. Coli solution:

1. Sterile tooth pick
2. Sterile tweezer
3. Alu foil
4. Pressure cooker/kitchen oven.
5. Conical flask/bottle

For isolation of nucleic acid:

1. 1.5 ml centrifuge tube
2. Centrifuge test tube
3. Micropipette
4. Vortex machine
5. Syringe with 26 G needle
6. UV spectrophotometer.
7. Centrifuge machine

Working procedure:

    Step-1: Preparation of Luria broth: for 100 ml volume of Luria broth 5gm of yeast extract, 10gm of NaCl, 10gm Tryptophan and 100ml distilled water were taken in a bottle. Then it is autoclaved for 15 minutes.

    Step-2: Preparation of E. Coli solution: In this method we need a sterile toothpick, a sterile tweezer and a culture of E. coli. The toothpick was sterilized by wrapping it in a foil and placing it in an autoclave for 15 minutes, a pressure cooker at 15 lb for 15 minutes or kitchen oven at 350F for 15 minutes. A match is used to flame the end of the pair of tweezer.

    The sterile toothpick was picked up with the sterile tweezer; a colony or section of E. coli was put on the toothpick and dropped into the bottle of L.B. The bottle was capped loosely and incubated at 370c.

    Step-3: Isolation of nucleic acid from E. Coli:

1. 3ml of overnight cultured was transferred to a 3ml centrifuge tube. Centrifuge for 3 minutes, then decanted the supernatant.
2. Re suspended cells in 100μL TE by vortexing and added 50 μL 10% SDS, 100-200μL 10N NaOH.
3. Sheared DNA by 3-5 passes through a 26G needle.
4. Extracted with 200-300μL phenol: CHCl3 (1:1) and shaken. After that supernatant was taken into another microfuge tube.
5. Precipitated nucleic acids by adding 50μL, 5M NaCl and 500μL 95% ethanol, vortexed and then took rest at – 200c for 30minutes, centrifuged for 10 minutes, decanted supernatant dry pellet.
6. Pellet was resuspended in 100μL TE buffer solution.
7. Determine the DNA concentration by measuring the absorbance at 260nm. (1 OD260 = 50μg/ml). For this 30 μL sample solution was taken into absorbance cell which was diluted with 2970 ml of distilled water.

Dilution Factor: It is the ratio between total amount of solution and amount of sample solution.

Here,    μL sample solution mixed with        μL distilled water and total amount of solution is (               ) μL =              μL

So, Dilution Factor =                   = 100

Calculation:

    The absorbance data was as follows-

    Blank solution =

    Sample with distilled water =

    Sample =         =

We know, 1 OD260 = 50μg/ml

Now Dilution Factor =         =

So, concentration of nucleic acid,

= Absorbance × 50 × Dilution factor

=

=              μg/ml.

Result:

    The concentration of nucleic acid is                     μg/ml.

Experiment No.  03	Date:
Name of the Experiment: Separation and visualization of DNA through agarose gel electrophoresis.

Principle:

    Electrophoresis is a separating technique for separating molecule in a mixture under the influence of applied electrical field. Normally it is used for separation of protein or nucleic acid. Agarose gel is common media for the electrophoresis of DNA. Agarose is a polysaccharide, which forms a solid gel when dissolved in aqueous solution; a concentration between 0.5-2% w/v agarose gel is submerged in the buffer solution with the sample wall side toward negative pole. The sample is dispensed into the wall with a micropipette and a current from the power supply is applied to the system. Since nucleic acids have a negative charge at pH of about 8. They will migrate within the agarose gel matrix from the negative to the positive pole. The agarose gel serves as a molecular sieve to separate molecules of DNA based on their size and the total charge of the molecule. DNA solution are normally loaded along with a colored dye (Bromophenol blue) so that the electrophoretic migration of the DNA through the gel can be visualized and stopped before the DNA migrate out of the gel and into the buffer solution.

    The migrating rate of charged macromolecules in a applied electric field is proportional of the total negative charge of the macromolecule and inversely proportional to its mass, small fragments of DNA migrate faster than larger ones.

    Finally, DNA molecules are invisible to the naked eye but can be seen in gel by staining them with a solution of a dye called ethidium bromide when excited with ultraviolet (UV) light.

Apparatus:

1. Electrophoresis Chamber/Equipment
2. Microwave oven
3. Micropipette
4. Eppendrof pipette
5. UV light box
6. Camera
7. Conical flask
8. Beaker
9. Volumetric flask

Reagent:

1. Agarose powder
2. 50 X TAE (Tris-acetate EDTA).
3. Ethidium bromide (5mg/ml or 10mg/ml).
4. Gel loading dye (50mM EDTA, 0.2% SDS, 50% glycerol and 0.05% w/v Bromophenol blue).

Procedure:

1. At first preparation of 500 ml 1X TAE from prepared 50 X TAE solution by simply diluting the stock solution by 50 X in deionized water. The buffer is now ready for use in running an agarose gel and in the preparation of agarose gel.
2. For the preparation of 1% gel, 0.5gm of agarose is added to 50ml 1X TAE.
3. Now heating is done to the solution to boiling in microwave oven to dissolve the agarose. For 50ml, it should take about 1 minute at power level 10.
4. Now putting the two dams into the slots on each side of the gel plate and made sure that they fit fight. After sure melted agarose is poured on to the gel plate in the electrophoresis box. Next the comb is placed in plate. The comb, with larger teeth is used when fewer sample is present.
5. The gel is cooled to room temperature and it should look cloudy. Then the dams and the comb are carefully removed. The buffer reservoir is filled with 1X TAE buffer until the buffer is 1-2 mm deep over the gel. If we add much more buffer it will comes in contact with the electrical contacts. The more buffer in the chamber, the higher the current will be when the gel run.
6. For the larger wells and smaller wells 10μL sample and 7μL sample is prepared respectively. Then 3μL of the loading dye is added to the sample. The samples is placed into adjacent walls, by using an Eppendrof pipette and a steady hand.
7. Electrophoresis of the sample is done at 100V for 45 minutes or until that has migrated at least 6 cm.
8. After turning the power off, the gel is removed from gel box and submerges it in the ethidium bromide staining solution (TAE X 1 + 6μl Ethidium solution) and shaking the gel for few minutes.
9. By wearing gloves, the gel is removed carefully from box and put it on to the UV light box and take a look on to the gel if ok, then a picture is taken.

Precaution:   

1. When working with ethidium bromide it is necessary to use gloves because it is a powerful mutagen.
2. UV protective glasses or cover the light box with the UV protective shield is necessary when the UV light is on because UV can produce skin cancer.
3. The comb should be pulled after 15 to 20 minutes but not quickly. It should be noticed that the comb is nearest to the back electrode (cathode).

10% SDS

Sodium Laurel Sulfate or Sodium Dodecyl:

    Stock Solution   

SDS (Electrophoresis-grade)         3.12 gm

Distilled H.O _                80 ml

*Total Volume:                 100 ml

After 80 ml of dH2O is added – stir, then fill up to 100 ml. Do not autoclave.

Sodium hydroxide (10N NaOH)

Dissolve 20 gm NaOH in 100 ml DW

NaOH	10	gram (g)
ddH20 to	25	milliliter (ml)
Total volume	35	milliliter (ml)

Phenol: Chloroform (1:1)

Weigh out 20g phenol (CAUTION-TOXIC AND CORROSIVE, WEAR GLOVES) in a glass beaker. Add 20 ml chloroform covr with clingfilm and mix well over a period of a

TE

	100 ml
10mM tris pH7.5(25oC)	1	ml of 1M
0.1mM EDTA	50µl of 0.2M (pH8.5)

TE Buffer (10:1)

1 M Tris-HCI (pH 8.0)	1	millilitre (ml)
0.5 M EDTA	0.2	millilitre (ml)
Water to	100	millilitre (ml)
Total volume	100	millilitre (ml)

TE Buffer (100:1)

1 M Tris-HCI (pH 8.0)	1	millilitre (ml)
0.5 M EDTA	0.02	millilitre (ml)
Water to	100	millilitre (ml)
Total volume	100	millilitre (ml)

Tris-HCI (1M)

Tris base	121.1	gram (g)
ddH20 to	100	millilitre (ml)
Total volume	100	millilitre (ml)

Note: dissolve in 800 ml of ddH20, addust to desired pH with concentrated HCI and add ddH20 to 1 litre

Sodium choloride (5M)

NaC1	292.2	gram (g)
ddH20 to	100	millilitre (ml)
Total volume	100	millilitre (ml)

Note: autoclave to sterilize

LB medium

Bacto-Tryptone	10	gram (g)
Bacto-yeast extract	5	gram (g)
NaC1	10	gram (g)
ddh20 to	1	litre (l)
Total volume	1	litre (l)

Note: adjust pH to 7.0 and autoclave to sterilize

EDTA (0.5M pH8.0)

Na2EDTA	186.1	gram (g)
ddH20 to	500	millilitre (ml)
Total volume	500	millilitre (ml)

Note: Dissolve in approx. 400 ml ddH20, adjust pH to 8.0 with 10 N NaOH, and adjust to 1 liter final volume

Page = 18

Photocopy = 18*1.35= 24.3

Binding = 15

Printing = 39

Total  =  39 (+ printing)

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