Various techniques in molecular bology

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techniques in molecular biology and cell line culturing with animal handelling

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  • REQUIREMENTS:EXTRACTION BUFFER10 mM Triscl (pH 8.0)0.1 mM EDTA (pH 8.0)20 µgm/ml Pancreatic RNase0.5% SDS PBSNacl - 0.8 gmKcl – 0.2 gmKH2PO4– 0.24 gmNa2HPO4 – 1.4 gmDissolve in 800 ml DW maintain the pH at 7.4 with Hcl and autoclave the solution.chloroform : isoamyl alcohols (24:1) make 10 ml in volume.Saturated phenol : chloroform : isoamyl alcohol (25:24:1) Take 5 ml of mixture no.3 and add 5 ml of saturated phenol.Procedure:sample is taken from the patient and stored in a vial coated with the EDTA(anti-coagulant)From the sample 500 µl blood is pipette out and mixed with the 500 µl PBS.The sample is then centrifuged at 5000 g for 10 min at 4°C.The supernatant is taken out and pellet is mixed with 200 µl extraction buffer and 1 µl RNase, This is then incubated at 37°C for 1 hr. 1 µl of proteinase-k is added and then it is incubated at 50°C for 3 hr.Now take the supernatant and add 200 µl of EB and 600 µl of saturated phenol and then it is centrifuged at 12000 rpm for 10 min.Step no. 6 was repeated twice without the EB and then supernatant is taken in fresh microcentrifuge tube.600 µl of chloroform : isoamyl alcohols (24:1) is added in the supernatant and it is centrifuged at 12000 rpm for 10 min, this step is repeated thrice.Supernatant is taken in fresh microcentrifuge tube and mixed with 600 µl of Saturated phenol : chloroform : isoamyl alcohol (25:24:1) then it is centrifuged at 12000 rpm for 10 min, this step is repeated twice.Now the supernatant obtained after step 9 is mixed with 60 µl of ammonium acetate and left overnight at -20°C for precipitation.After precipitation it is centrifuged at 12000 rpm for 10 min at 4 °C.Now for washing supernatant is discarded and the pellet is mixed with 70 % ethanol and centrifuge at 12000 rpm for 10 min and then tubes were decanted and kept on RT for drying for 4 hr.After tubes are completely dried the pellet is dissolved in the 30 µl of TE buffer and incubated for 2 hr at 37°C. visualisation:For visualising the DNA we prepared 25 ml of 0.8% agar gel and casted it in the electrophoric unit.To load in the wells 10 µl of sample is mixed with the 3 µl gel loading dye and run at 75 mV for 1.5 hrs.The bands are visualized using UV transilluminator.
  • HRP (horse radish peroxidase)
  • Various techniques in molecular bology

    1. 1. DNA
    2. 2.  Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses.  Along with RNA and proteins, DNA is one of the three major macromolecules essential for all known forms of life.
    3. 3. Isolation of DNA from human blood  Human blood contains several types of cells such as RBC’s and WBC’s these are present in blood in different amount according to their requirement and in this we can isolate DNA from the WBC’s as they contains nucleus while prior one does not.
    4. 4. REQUIREMENTS:  a. b. c. d. EXTRACTION BUFFER 10 mM Triscl (pH 8.0) 0.1 mM EDTA (pH 8.0) 20 µgm/ml Pancreatic RNase 0.5% SDS PBS Nacl - 0.8 gm Kcl – 0.2 gm KH2PO4 – 0.24 gm Na2HPO4 – 1.4 gm Dissolve in 800 ml DW maintain the pH at 7.4 with Hcl and autoclave the solution.  chloroform : isoamyl alcohol (24:1) make 10 ml in volume.  Saturated phenol : chloroform : isoamyl alcohol (25:24:1) Take 5 ml of mixture no.3 and add 5 ml of saturated phenol.  a. b. c. d. 
    5. 5. Procedure  Sample is taken from the patient and stored in a vial coated with the EDTA(anti-coagulant)  From the sample 500 µl blood is pipette out and mixed with the 500 µl PBS.  The sample is then centrifuged at 5000 g for 10 min at 4°C.  The supernatant is taken out and pellet is mixed with 200 µl extraction buffer and 1 µl RNase, This is then incubated at 37°C for 1 hr.
    6. 6.  add 200 µl of EB and 600 µl of saturated phenol in Aq. Layer and centrifuge it at 12000 rpm for 10 min.  Step no. 6 was repeated twice without the EB and then supernatant is taken in fresh microcentrifuge tube.  600 µl of chloroform : isoamyl alcohol (24:1) is added in the supernatant and it is centrifuged at 12000 rpm for 10 min, this step is repeated thrice.  Supernatant is taken in fresh microcentrifuge tube and mixed with 600 µl of Saturated phenol : chloroform : isoamyl alcohol (25:24:1) then it is centrifuged at 12000 rpm for 10 min, this step is repeated twice.  Now the supernatant is mixed with 60 µl of ammonium acetate and left overnight at -20°C for precipitation.
    7. 7.  After precipitation it is centrifuged at 12000 rpm for 10 min at 4 °C.  1 µl of proteinase-k is added and then it is incubated at 50°C for 3 hr.  Now for washing supernatant is discarded and the pellet is mixed with 70 % ethanol and centrifuge at 12000 rpm for 10 min and then tubes were decanted and kept on RT for drying for 4 hr.  After tubes are completely dried the pellet is dissolved in the 30 µl of TE buffer and incubated for 2 hr at 37°C.
    8. 8. Visualisation  For visualising the DNA we prepared 25 ml of 0.8% agar gel and casted it in the electrophoretic unit.  To load in the wells 10 µl of sample is mixed with the 3 µl gel loading dye and run at 75 mV for 1.5 hrs.  The bands are transilluminator. visualized using UV
    9. 9. DNA
    10. 10.  It is done to check the mutation present in the DNA sample, and to identify the mutation sites we have to use a particular restriction enzyme as in BRaf gene which is present in MAP kinase pathway V600E mutation is very common in papillary thyroid carcinoma.  If the mutation has taken place then the site for the RE will be depleted in the DNA and it will give single band but if it is having no mutation then this will result in the three bands as site for RE will be present.
    11. 11. REQUIREMENTS 2 µl of buffer (tango)  10 µl of DNA sample  1 µl of restriction enzyme  7 µl of RNase free water Mix these to make the total volume 20 µl and take in microcentrifuge tubes  25 ml of 3% agrose gel with 4 µl of ETBr in 1X TBE.  Molecular ladder: gene ruler 50 bp (1 µg/ µl). 
    12. 12. PROCEDURE  The prepared 20 µl solution incubated for overnight.  Then cast the gel in the electrophoretic unit and pre run it for 5 min for at 20 mV.  Load the 8 µl of incubated solution with 2 µl of gel loading dye.
    13. 13.  Also load the 5 µl of ladder then run it at 70 mV for 2.5 hrs.  Now the gel is observed in the UV transilluminator.
    14. 14. Samples 1,2,3,4 237 bp 117 bp 87 bp 33 bp ladder
    15. 15. DNA
    16. 16.  This technique can be used for many applications but mainly it is used for the amplification of DNA.  It uses polymerase enzyme and primers to replicate the desired DNA sequence.
    17. 17. REQUIREMENTS Microtiter plate  PCR kit  PCR machine 
    18. 18. PROCEDURE  The sample is taken in the plate provided with the machine.  A control sample is also used generally some housekeeping gene like betaactin.  Now the primers are added and the polymerase enzyme too.
    19. 19.  Load the plate in the machine and set the plate profile in the software.  Now after the process is finished retrieve the plate with amplified sample.
    20. 20. VISUALISATION  3% agrose gel.  PCR products are loaded in the wells with the gel loading dye.  A ladder is also used for conformation of the size of product.
    21. 21. Products of PCR Primer 1,2,3,4,5,6,7 Ladder
    22. 22. RNA
    23. 23. RT-PCR THIS IS A TECHNIQUE THROUGH WHICH WE CAN AMPLIFY THE SELECTED cDNA STRANDS WITHIN A VERY SHORT TIME SPAN. IN THIS WE CAN VISUALIZE THE AMPLIFICATION IN REAL TIME WITH THE HELP OF MACHINE.
    24. 24. REQUIREMENTS  cDNA  rt-PCR kit  PCR well plate  Trizol (phenol + guanidine thiocyanate)  Tissue sample  Homogenizer  Sybr green master mix
    25. 25. RNA EXTRACTION( TRIZOL METHOD)  Homogenization: tissue preserved in liq. N2 is homogenized in Trizol reagent.  Phase separation: Incubate for 5 min then add 200 µl chloroform/1ml Trizol incubate for 3 min then centrifuge at 12000g for 15 min at 4 C.  Upper aq. Phase contains RNA.  1-bromo-3-chloropropane.
    26. 26.  RNA precipitation: Mix isopropyl alcohol 0.5 ml/1ml Trizol Centrifuge at 12000g for 10 min at 4°C  RNA wash: mix pellet with 1ml ethanol (70%)/1ml Trizol Centrifuge at 7500g for 5 min at 4 C
    27. 27.  Resolving the RNA: Dry the pellet Dissolve in 50µl RNase free water Store at -80 °C
    28. 28. Convert the RNA into cDNA Take 500 ng to 2µg of the converted sample Add in rtPCR plate along with Sybr green master mix and distill water Start the program in machine and then see the amplification in real time Obtain the amplified sample
    29. 29. TPI
    30. 30.  It is the building block of our body. It is present in tremendous variety and play several roles altogether in our body. And to study any change in any organism’s body we can go for checking the protein’s nature and amount. To check the level of expression in the body of any organism we can opt for following techniques:  Western blotting.  Immunohisto chemistry.
    31. 31. PROTEIN
    32. 32.  This technique is used for the separation of proteins on the basis of their molecular weight.  level of expression with the help of any reaction like Ag-Ab interaction and developing the blot on x ray film with the help of any chromogenic substrate.
    33. 33.  Gel casting unit  Chemicals for the PAG, separating gel will be of 6-15%  Running buffer (pH 8.3)  Transferring buffer (pH 8.5)  Nitrocellulose membrane  Transfer unit  Current supply unit
    34. 34.  Prepare the unit and buffers.  Now put the unit in the electrophoretic tank and add running buffer.  Load the equal amount of the protein samples (in micrograms) with loading dye.  Attach the current supply unit and let it run until dye has reached the bottom.
    35. 35.  On completion remove the gel and assemble the transfer unit and transfer the proteins on nitrocellulose membrane with help of transfer buffer.  After transfer has completed take the membrane and use ponctuS dye to stain the proteins, it is not permanent hence after visualisation it can be washed.  Prepare 5% blocking solution (milk) in TBST/PBST, and give exposure for 1 hr.  We prepared the primary antibody in 1-5%BSA diluent and add to the membrane. This process will take place over night in the cold room at 4°C.
    36. 36.  Next day the membrane is again washed with the TBST three times for at RT for 15 min each.  The membrane is washed using TBS solution on Rocking platform. Three washes of 15 min each will be given.  Then secondary antibody is diluted in the blocking solution and added to the membrane and the exposure given will be of 1 hr.  After that again washing with the TBST three times and then blot development procedure.
    37. 37.  In Blot development we take the membrane out and add the substrate which is having luxogenic reaction.  The time of reaction taking place is moderated according to the individual and after the exposure of X-ray film, it will be put in the machine for development of blot.  The intensity of the blot is proportional to the level of expression.  Luminol+H2O2 is the substrate.
    38. 38. PROTEIN
    39. 39.  This technique is used for checking the localisation of any particular protein at a particular time in the cell. can’t quantify the protein hence generally done parallel with the western blotting. It is very important as it can show the effect of any disease or any compound on a particular protein.  It
    40. 40.                Prepared blocks of desired tissue Water bath Poly lysine coated slides. Xylene Alcohol (100%, 90%, 70%, 50%) PBS Citrate buffer Na2BH4 H2O2 Blocking solution (5% sheep serum, 0.1% triton) DPX Primary antibody HRP labelled Secondary antibody Streptavidin-biotin complex Pressure cooker
    41. 41.  Place the block on the mount and align it to the blade.  Adjust the section size and remove the extra wax.  Switch ON the water bath, and start cutting the sections.  Now the section will be obtained and we can fix them on the polylysin coated slides.  Deparaffinised on hot plate at 60°C.
    42. 42.  The slides are placed in the coupling jar with Xylene and jars are put into the hot air oven at 60°C. After 10 minutes use new Xylene jar.  Then rehydration is done by keeping the slides into the alcohol and slowly decreasing the concentration of the alcohol at last it will be washed into the distil water for 5 min.  Then min. the slides will be transferred into PBS for 5
    43. 43.  Then transfer the slides into the citrate buffer and transfer the jar into a pressure cooker with water at 120°C this will be kept for about 45 min.  Now the slides were cooled down and then transferred to the jar with PBS for three wash10 min each.  1% Sodium borohydrate (Na2BH4) is prepared and the exposure to the slides is given in the dark.  Again wash the slides with PBS two times 10 min each.  Then we prepare the blocking solution add it to the slides and keep the slides covered with parafilm.  After this we will give the exposure of primary antibody (1:500) and keep them covered for overnight in medicool at 4°C.
    44. 44.  Next day after bringing the slides at RT we wash them with the PBS three times 5 min each.  Then slides are transferred in 3%H2O2 freshly prepared for 20 min.  Then again wash with PBS three times 10 min each.  Take out the slides on the slide chamber and expose them to the secondary antibody with sheep serum and 1X PBS for 60 min.  Again the slides are washed with PBS three times 10 min each.
    45. 45.  Now take out the slides and add the substrate for 15 min.  Wash with PBS three times each of 5 min.  Now the exposure of DAB is given to intensify the signal for 20 min.  Wash with PBS and after drying mount with DPX this increases the refractive index.  Now seal them with transparent paint and leave them for drying.  When completely dried observe under the Phase Contrast Microscope. This will show the specified protein.
    46. 46. Antigen Aquaporin II in kidney cell through phase contrast microscope at 10X
    47. 47. Antigen Aquaporin II in kidney cell through phase contrast microscope at 40X
    48. 48. PROTEIN
    49. 49.  It is done to obtain the section of any tissue from any organ of an organism.  This block is used in immunohistochemistry.
    50. 50.  Select out the subject animal and sedate it with the chloroform.  Then cut out its head and retrieve the brain with the help of dissection kit and store it in the PFA.  After 24 hrs we go for dehydration of the tissue.
    51. 51. 50% alcohol 1.5hrs 70% alcohol 1.5hrs 80% alcohol 1.5hrs 90% alcohol 1.5hrs 100% alcohol 1.5hrs 100% alcohol 1.5hrs 100% alcohol 1.5hrs or over night
    52. 52. After last step we leave it for the night and then use chloroform to replace the alcohol chloroform 1.5hrs chloroform 1.5hrs chloroform 1.5hrs
    53. 53. We use molten wax to totally occupy the free spaces within the tissue so that it retains its structure. Wax 1.5hrs Wax 1.5hrs Wax 1.5hrs
    54. 54.  After the final exposure transfer the tissue at the base of mould and pour the molten wax on it then set on the cold plate and cover the mould with the casket.  After 4°C. it solidifies remove the mould and store at
    55. 55. BRAIN TISSUE FROM MICE
    56. 56. PROTEIN ESTIMATION (BRADFORD METHOD)
    57. 57.  Estimation of protein content in any sample provided either by patient or obtained through animal model tissue is important as according to the concentration present we will be able to identify the protein’s molecular weight and use it in western blotting.
    58. 58.  Protein standard sample  PBS  Bradford dye  Spectrophotometer  Protein samples
    59. 59.  First we prepared a working stock solution of 100 µg/ml from 1.34 µg/ml.  From this working stock we prepared standards of different concentration:  0.8 µg/ml  1.6 µg/ml  4.0 µg/ml  6.0 µg/ml  12.0 µg/ml 20 µl WS + 1980 PBS 40 µl WS + 1960 PBS 100 µl WS + 1900 PBS 150 µl WS + 1850 PBS 300 µl WS + 1700 PBS
    60. 60.  Now we mixed 200 µl dye with 800 µl of all these standards and also prepared blank by taking 800 µl PBS in place of standards.  The samples which are to be examined are taken 800 µl and mixed with 200 µl of dye.  Now we started taking readings from UV spectrophotometer.  Thus obtained readings will give a plot of conc vs absorbance. Thus we can calculate the concentration of unknown sample.
    61. 61. 0.6 0.5 0.4 abs 0.3 abs 0.2 0.1 0 0 2 4 6 concentration 8 10 12
    62. 62. PRIMARY CULTURE
    63. 63.  This technique is important to study the behaviour of cells towards a specific condition or compound.  The tissue used in this is either obtained through model organism or biopsy.
    64. 64.  Model organism  Dissection kit  PBS  Media (DMEM) with phenol red  Serum (calf serum)  Filter assembly  Sterilised glass wares  6 well plate
    65. 65.  Put the serum for freeze thaw and after that filter sterilize it.  Dissolve the media in milliQ water and then filter sterilize it.  Model organism is dissected and its desired organ is retrieved (in this case kidney).  The organ is washed properly in PBS, bring it in culture room.
    66. 66.  Neatly with the help of surgical blade cleave the kidney transversely.  Retrieve the cortex part of the kidney and discard the remaining part.  Take the tissues in PBS and prepare homogenate and through repetitive pipetting make single cells.  Equal volume of cells is added in the wells.
    67. 67.  Now the media is added in the cells and then serum is also added.  The cells are daily washed with PBS and media is changed.  To observe the growth of cells inverted light microscope is used.  After it has taken up more than 80% volume of the wells we can passage the cells.
    68. 68. visualisation Kidney cells through inverted miroscope
    69. 69.  This technique is important to study the cell on sub cellular level.  We can visualise these and obtain information regarding the cells.  We check the level of any specific protein at any specific location.
    70. 70.  Model organism  Mitochondrial buffer  centrifuge Mitochondrial buffer  1mM EDTA(pH 8.0)  0.32 M sucrose  10 mM Tris-cl(pH 7.4)  1mM PMSF  1mM DTT
    71. 71.  Dissect the model organism and retrieve the desired tissue in this case cerebral cortex and Preserve it in the liquid nitrogen.  Protease inhibitor cocktail 5 µl/ml buffer, weigh 100 mg tissue and take it in 1ml mitochondrial buffer.  Homogenize in power homogenizer then keep on ice for 15 min.
    72. 72.  Centrifuge at 700 rpm for 10 min and take out the supernatant.  The nuclei and unbroken cells were pellet down by centrifuging at 2000 rpm for 10 min.  Thus obtained pellet is nuclear fraction (A) add buffer and store at -20 °C.  The obtained supernatant is centrifuged at 2000 rpm for 10 min.  Thus obtained pellet is nuclear fraction (B) add buffer and store at -20 °C.
    73. 73.  The obtained supernatant is centrifuged at 2000 rpm for 15 min.  Thus obtained pellet is mitochondrial fraction (C) add buffer and store at -20 °C.  The obtained supernatant is centrifuged at 2000 rpm for 15 min.  Thus obtained pellet is mitochondrial fraction (D) add buffer and store at -20 °C.  The remaining supernatant is designated as cytosolic fraction (E), add buffer and store at -20 °C.
    74. 74.  This procedure is very important as it prepares the model organisms on which experiment models can be executed. These are kept in a very specific condition and given specific diet so that their growth and development pattern can be controlled.  On the basis of desired condition of these organisms we prepare a model according to which they are gradually brought to that condition and then their condition is analysed by various tests.
    75. 75.  These are having a life span of about 1 year but their various growth stages are required hence breeding is required and it is also monitored.  For various experimental models like induced nephropathy(diabetic), hyperthyroidism and hypothyroidism different set of diet and growth stages are required hence they are grown separately.
    76. 76.  Condition of parents is monitored constantly and their diet is also regulated according to the model.  Every morning we have to check the urinary sample for sign of fertilisation if sperm is found then these are separated and time is noted down.  Thus different embryonic stages can be obtained and we can check for the level of effect of any particular exposure that was given to the parents.
    77. 77.  To preserve adult organism’s organs and serum we have to set a different model involving different kind of parameters.  Their diet is kept regulated and also we have to set a control organism to compare the growth.  Daily changing of bedding and sanitation is important as we don’t want to affect the environmental condition.  Giving daily set dosage of medicine is also very crucial.
    78. 78.  After completing growth model we have to procure the organs and blood from that organism and after that we preserve organs either in liquid nitrogen(for western blotting) or in PFA(block preparation and IHC).  For taking out the blood properly we have to keep the organism alive but sedated so that his heart keeps pumping and we would be able to extract the blood and from that we can isolate serum.
    79. 79.  If we have to preserve bones (vertebrae, femur) to check the effect of Osteocalcin and RNA analysis we also preserve them in PFA.  Different embryonic stages are marked as E16, E18 according to their growth stage.  These are nocturnal hence are kept in dark and quite room.  If we bring them out from dark room their hormonal level will change so before going for dissection we have to let them stabilize.
    80. 80. Embryo of 18 days
    81. 81.  If we are inducing any medical condition in the organism then we have to closely monitor the changes in the health of the subject. Like in case of diabetic condition we have to check for blood glucose level.  To check their level of muscular activity we use ferries wheel and thus we can find out which subject is doing good and whose condition is going bad.  Proper discarding of the dissected subject is also important.  Always use sterile kit for dissection and keep all the necessities ready.
    82. 82. THANK YOU

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