basic techniques of biotechnology


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The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.

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basic techniques of biotechnology

  1. 1. Basic Techniques Of Biotechnology By:- Shalini Kaushik B.Tech (BT) 4th year
  2. 2. CONTENTS • Isolation Of genomic DNA from Bacteria. • Visualization of isolated DNA through Agarose gel Electrophoresis. • Polymerase Chain Reaction (PCR). • SDS- PAGE • Restriction Digestion • Nano particle synthesis & to test their antimicrobial activity. • DOT-ELISA. • Plant tissue culture. • Cultivation of Spirulina platensis by using zarrok`s media .
  4. 4. CONCENTRATION OF CHEMICALS USED IN DNA EXTRACTION • TE-Buffer : 10mM tris-cl (pH- 8.0) 1mM EDTA (pH- 8.0) • Lysis buffer (10ml):9.34ml TE-Buffer 600 µl of 10% SDS 60 µl proteinase k(20mg/ml) Overall:: TE Buffer(20ml)= .0242gm Tris-Cl +.00744gm EDTA. Lysis Buffer(10ml)=9.34ml TE-Buffer+.06gm SDS+ 12mg proteinase k.
  5. 5. E.Coli DNA EXTRACTION Pellet of E.coli culture is added with lysis buffer. Lysis Buffer = TE Buffer+ SDS+ proteinase k LYSE THE CELL TrisHCl AND EDTA Denat ure the structure of protei -n Remove out protei n
  6. 6. CONT…. • After incubation add chloroform and isoamyl alcohol instead of phenol. • Three layers are formed: AQUOS LAYER INTERMEDIATE LAYER BOTTOM LAYER having RNA AND DNA having DENATURED PROTEIN having CHLOROFORM AND ISOAMYL ALCOHOL
  7. 7. CONT…. • After taking aqueous layer in new vial and then centrifuge. Pellet is having RNA so take supernatant. • Add ethanol in supernatant and incubate. • Centrifuge and take pellet. • Air dry the pellet and store by adding TE-buffer.
  8. 8. STEPS OF EXTRACTION OF DNA FROM A E.coli CELL centrifuge supernatant pellet 1.5ml E.coli culture Vortex and incubate at 37 degree centigrade for 1 hr. 600l lysis buffer PELLET Add chloroform and isoamyl alcohol
  9. 9. Take aq.layer and centrifuge Incubate at 20 degree cent. For 30 min. centrifuge take supernatant Add 2.5µl ethanol
  10. 10. Take pellet and add 1ml 70% ethanol centrifuge Take pellet and air dry it Add TE Buffer and incubate
  12. 12. INTRODUCTION • Separation of DNA,RNA,proteins by applying an electric field to move negatively charged molecule through an agarose matrix. • Molecules separated in their fragments on the basis of their size by sieving. • It is used to 1.Separate proteins. 2.Separate mix population of DNA. 3.Separate RNA fragment by length. 4.Estimate size of DNA and RNA.
  13. 13. COMPOSITION OF AGAROSE GEL AND BUFFER USED • AGAROSE GEL-: 1% of 100ml is used. means .5g agarose in 49ml d.w and 1ml 50x buffer. 5 µl EtBr is used. • 600ml BUFFER-: (Tank Buffer) 12ml 50x buffer + 588ml d.w.
  14. 14. PROCESS OF GEL ELECTROPHORESIS • Prepared agarose gel at 45ºC was poured in gel casting plate where comb was already placed. • After formation of gel, comb was removed and wells formed. • Buffer was poured in the tank. • Sample along with dye was loaded to the well. Marker was also added in another well. • Apply voltage and under the influence of the electric field, movement starts.
  15. 15. ROLE OF EtBr Role of EtBr it is a fluorescent dye which fluorescent after intercalating between two strands , under a UV light. It emit a particular wavelength of light which comes under visible light.
  18. 18. POLYMERASE CHAIN REACTION • Given by Kary mullis in 1983. • Biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA. • PRINCIPLE - based on DNA polymerization reaction. • Thermal cycling consisting of repeated cycles of heating and cooling of the reaction for the DNA melting and enzymatic replication of DNA using Taq polymerase and primer sequence.
  19. 19. REQUIREMENT FOR PCR REACTION: • • • • • • • Nuclease free water : 18.5 µl or 37 µl 10x Taq pol. Assay buffer : 2.5 µl or 5 µl dNTPs : 2 µl or 1 µl Forward primer : 2 µl or 1 µl Reverse primer : 2 µl or 1 µl Extracted template DNA of E.coli :1 µl or 0.5 µl Taq polymerase : 1 µl or 0.5 µl TOTAL :: 50 µl or 25 µl
  20. 20. TO AMPLIFY 1Kb FRAGMENT FROM EXTRACTED TEMPLATE DNA FROM E.coli 1 step 2 step 3 step 4step Initial denatur annea extent final final Denaturation -ation -ling -ion extent hold 94ºC 94ºC 60ºC 72ºC 72ºC 4ºC 2 min. 30sec. 30sec. 1min. 2min. 60min. 32 cycles
  21. 21. GRAPH SHOWING PCR REACTION BETWEEN TIME AND TEMPRETURE .T E M P R E T U R E 94 2min 94 30sec 72 72 1min 2min in ºC 60 30se c 4 60min TIME
  22. 22. REACTION INVOLVED IN PCR 3’ 5’ . 1st 5’ 3’ CYCLE DENATURATION 3’ 94ºC 5’ 5’ 3’ 60ºC ANNEALING 5’ 3’ 3’ 5’ 5’ 5’ EXTENTION 72ºC
  23. 23. 3’ 5’ . 5’ 5’ 3’ 3’ 3’ 5’ DENATURATION 5’ 3’ 5’ 3’ 5’ 2nd CYCLE 3’ 3’ 5’ ANNEALING 3’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 5’
  24. 24. . EXTENTION 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 5’ 3’ 3’ FORMATION OF 8 DNA STRANDS 3’ 5’
  25. 25. RESULT OF PCR AFTER ELECTROFORESIS Resulted DNA band of 1kb
  26. 26. SDS PAGE
  27. 27. PRINCIPLE OF SDS PAGE Principle is based on the separation of protein on the basis of their size and their charge. SDS applied to protein sample to impart a negative charge linearize the protein. Electric field applied across the gel, causing the negative charged proteins to migrate across the gel towards positive electrode. SDS-PAGE is chemically inert and produce different pore size. NOTE: There is a discontinuous buffer system.
  28. 28. CONT…. Velocity of charged particles moving in electric field is -Directly proportional to the field strength and charge on molecule -Inversely proportional to the size and viscosity of molecule.
  29. 29. SAMPLE PREPARATION STACKING GEL AND RESOLVING GEL : CHEMICALS 1. Acrylamide/ Bisacrylamide 2. Gel buffer (tris-HCl-:1.5M) 3. d.water 4. SDS (10%) 5. TEMED 6. APS (20%) CONCENTRATION Stacking gel .5ml 0.63ml (pH-:6.8) 1.32ml 25 µl 1 µl 25 µl Resolving gel 1.98ml 1.25ml (pH-:8.8) 1.77ml 50 µl 4 µl 38 µl
  30. 30. CONT….. • ELECTRODE BUFFER : Tris-HCl 3gm Glysin 14.4gm SDS 1gm • SAMPLE : 30 µl protein sample + 30 µl loading dye. • MARKER : 10 µl • DYES : Staining dye distaining dye .8gm coomassie blue d.w-:630ml in 1l d.w glacial acid-:70ml methanol-:300ml
  31. 31. CHEMICAL INGRADIENTS AND THEIR ROLES • Components of loading dye:-It is colorless progress through the gel. It is anionic of known electrophoric mobility. Move ahead protein. 1.Tris base-: maintain Ph. 2.BME( Beta mercapto ethanol)-:breaks disulfide bonds. 3.SDS-: linearize proteins and impart negative charge to proteins. 4.Glycerol-: increase density of is non-ionic and non-reactive toward proteins to interfering with electricity.
  32. 32. Cont.….. • Components of LGB and UGB buffer used:1. Acrylamide-:When dissolved in water autopolymerization of acrylamide takes place. Joining of molecules head to tail fashion to form single chain polymer. 2. Bisacrylamide-:Cross linking agent for polyacrylamide gel. Two acrylamide molecule coupled head to tail at their nonreactive ends. Hence cross linked two polyacrylamide chains to one another results to a gel formation. 3. TEMED (Tetramethylethylene diamine)-:provide free radicals. 4. APS (Ammonium per sulfate)-:stabilize free radicals and forms the gel.
  33. 33. Cont.…… • Components of electrode buffer:1. Tris-HCL-:When voltage applied. H+ ions and Cl- ions dissociates. Cl-ve ions are highly mobile as they are small in size as well as negatively charged. Hence it is always ahead than protein and glycine. 2. SDS-:It bound to the protein.(1.4gm SDS bound 1gm protein)and form SDS bound protein complex. Coats protein with uniform negative charge. 3. Glycine-:Weak acid which is neutral or protonated in the stacking gel while it becomes glycinate or deprotonated in the resolving gel.
  34. 34. Cont.…… • Size varies as:-Cl- < glycine < SDS bound protein NOTE: Glycine slows down in stacking gel do move but with less mobility than Cl- ions and protein. In resolving gel glycinate move behind Clbut ahead to proteins.
  35. 35. Cont.……… • Staining dye:- It is anionic, non-polar, nonspecifically bound to protein. Allowing visualization of separated protein. Different protein will appear as distinct bands within the gel. • Distaining dye:- It is used to destain the excessive dye.
  36. 36. WHY STACKING GEL IS 6% AND RESOLVING GEL IS 12% ? • Stacking gel is having less amount of acrylamide and bisacrylamide results in large pore size. Hence all proteins of different size stack in the same line ready to move from a same point. • Resolving gel have more amount of same due to which pore size is small. So proteins distinguish acc. to different size.
  37. 37. PROCESS OF SDS-PAGE • Prepared resolving gel was poured between two glass plates. • After that stacking gel was poured over it. • Then comb was placed between the two glass plates having gel between them. • Comb was removed and wells formed in which protein sample with tracking dye was loaded further.
  38. 38. DURING PROCESS Wells formed after Removing comb. (4 wells contain protein Sample along with dye And one well contain Marker) Dye tracking the Path of protein move -ment.
  39. 39. STAINING AND DESTAINING OF GEL • Staining dye is used to stain the proteins pathway. • After staining remove excessive stain by destaining dye. It will left for overnight. • After removing the excessive dye. Take the picture of bands of proteins onto the gel.
  40. 40. Cont.…….. For destaining the excessive dye, shaking is provided overnight with the help of decoloring shaker.
  43. 43. INTRODUCTION OF RESTRICTION ENDONUCLEASE • Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. • Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments.
  45. 45. PROCEDURE • Add these chemicals in two different vials. • Then we started electrophoresis. • In four different wells, we add In 1st well DNA digested with EcoR1(vial1) In 2nd well DNA digested with Hind III(vial2) In 3rd well standard DNA (undigested DNA) In 4th well digested DNA
  46. 46. RESULTS
  48. 48. INTRODUCTION OF NANOPARTICLES A nanometer is a billionth of a meter or 10-9 m. How small is nanometer? •If a baseball is the size of Earth, a nanoparticle would be the size of an apple. •We can also compare it with things in the natural world. Less than a nanometerIndi vidual atoms are up to a few tenths of a nanometer in diameter Nanometer Thousands of nanometers Ten shoulderto-shoulder Biological cells, hydrogen like these red atoms (blue blood cells, balls) span 1 have diameters nanometer. in the range of DNA molecules thousands of nanometers are about 2.5nm wide A million nanometers An ant is millions of nanometers across Billions of nanometers A two meter tall male is two billion nanometers tall
  49. 49. PROCEDURE OF EXTRACTION • 0.01gm AgNO3 dissolved in 100ml distilled water . AgNO3 dissociate after dissolving in water. AgNO3 Ag+ + NO3 - can be stabilized by adding bio resource (having large photo -synthetic activity)
  50. 50. Cont…….. • • • • Leaves of Moringa is taken as bioresource. Leaves were crushed and then centrifuge. Take supernatant . Add supernatant in .1gm AgNO3 in 100ml distilled water. • Kept onto the magnetic stirrer for overnight. • Nanoparticles formed by changing the colour of solution.
  52. 52. ANTIMICROBIAL ACTIVITY OF NANOPARTICLES PROCEDURE -: • Nutrient agar media was prepared for bacteria and PDA for fungi. • Media was autoclaved and poured in the petriplates. In NA, some plates was inoculated E.coli and some was inoculated with pseudomonas. • While in PDA, A.niger was inoculated. • 5 wells were made in a single plate in which 20µl,40µl,60µl, 80µl of nanoparticles were poured and ketoconazole was added in middle well in PDA plate and streptomycin in NA plates. • Overnight incubation was given.
  53. 53. RESULTS SHOWING ANTIMICROBIAL ACTIVITY OF NANOPARTICLES • FOR BACTERIA :Species concentration of nanoparticles E.coli 20 µl 40 µl 60 µl 80 µl diameter of inhibition zone 9mm 10mm 11mm 13mm
  54. 54. Cont.………. pseudomonas 20 µl 40 µl 60 µl 80 µl • FOR FUNGI: Sps. Conc. of nanoparticles A.niger 20 µl 40 µl 60 µl 80 µl 9mm 10.5mm 11mm 12.5mm diameter of zone of inhibition 7mm 9.5mm 10mm 11mm
  55. 55. RESULTS Petriplates Showing Antimicrobial Activity:- Presence of clear zone shows the inhibition of E.coli by silver nanoparticles
  56. 56. Cont…….. Presence of clear zone shows inhibition of pseudomonas by nanoparticles
  57. 57. Cont………. . Presence of clear zone around the wells indicates the inhibition of fungi (Aspergilus niger)
  59. 59. INTRODUCTION OF Dot - ELISA • Antigen is directly sandwiched between two antibodies which react with two different epitopes on the same antigen. • One of the antibodies is immobilized onto the solid support and second is linked to the enzyme. • Antigen present in the test sample is first linked to the immobilized antibody and then with the enzyme linked antibody. • Incubate the strip with an appropriate chromogenic substrate which is converted into colored and insoluble product.
  61. 61. PROCEDURE 1st . Dot-ELISA strip +1x assay buffer+ serum sample : sequence A sequence B -ve control Test zone +ve control zone INCUBATE FOR 20MIN., WASH sequence A sequence B No binding
  62. 62. Cont.………. 2nd. Add enzyme antibody conjugate(antibody-HRP) INCUBATE FOR 20 MIN. , WASH sequence A sequence B No binding with enzyme linked antibody Ab-HRP+ (Ag+strip(Ab))
  63. 63. Cont.……………. 3rd . Add substrate (TMB/H2O2): INCUBATE FOR 20 MIN. ,WASH Sequence A Sequence B No binding of Binding of the substrate. Substrate Hence no blue cause blue Spot in test zone spot. therefore, 2H2O2 HRP + O2 TMB 2H2O + O2 BLUE COLOUR
  65. 65. INTRODUCTION • Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. • Plant tissue culture is widely used to produce clones of a plant in a method known as micro propagation. • The production of multiples of plants is possible in the absence of seeds or necessary pollinators to produce seeds.
  66. 66. PROCEDURE • Nutritional medium was prepared by the addition of some macronutrients, micronutrients, vitamins& organics for the inoculation of seed for callus culture(pH-5.7). • Moong seeds are washed with the tap water for 2 times. • Now washed with the detergent. • Again washed with the tap water twice. • Now washed with the distilled water twice. • Then washed with the .1% HgCl2 for 1 min. • Now washed with the autoclaved distilled water for three times. • Now inoculate the seeds into the MS medium. • Flasks were kept in the growth chamber for proper growth.
  68. 68. Cultivation of Spirulina Platensis by using of Zarrok's media
  69. 69. INTRODUCTION SPIRULINA is a microscopic blue green algae in the shape of a spiral coil living in sea & fresh water. spirulina is the common name for human & animal food produced from two species of cynobacteria ; arthrospira platensis and arthrospira .though referred to as algae because they are aquatic organisms capable of photosynthesis. cynobacteria are not related to any of the various eukaryotic algae.
  70. 70. Composition of Zarrok’s Media NaHCo3 NaNo3 NaCl K2So4 K2HPo4 MgSo4.7H2OFeSo4.7H2OCaCl2.2H2O EDTA- 4.8gm/lt 2.5gm/lt 1gm/lt 1gm/lt 0.5gm/lt 0.2gm/lt 0.01gm/lt 0.04gm/lt 0.08gm/lt
  71. 71. PROCEDURE 1- Measured 50 ml of zarrouk`s media in one flask under laminar air flow. 2- culture of Spirulina was inoculated Platensis in 50 ml of zarrouk’s media under laminar . 3 - OD up to 0.3 by using spectrophotometer at 750 nm was adjusted. 4-after adjusting OD at 0.3, Spirulina culture was put in tissue culture laboratory . 5- suitable condition for culture was provided.
  72. 72. RESULTS After 9 days incubation culture was at harvesting stage.