basic techniques of biotechnology

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.

1. Basic Techniques Of Biotechnology By :- Shalini Kaushik B.Tech (BT) 4 th year

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 .

3. EXTRACTION OF E.coli GENOMIC DNA

4. TE-Buffer : 10mM tris-cl (pH- 8.0) 1mM EDTA (pH- 8.0) Lysis buffer (10ml):9.34ml TE-Buffer 600 µ l of 10% SDS 60 µ l 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. CONCENTRATION OF CHEMICALS USED IN DNA EXTRACTION

5. Pellet of E.coli culture is added with lysis buffer. Lysis Buffer = TE Buffer+ SDS+ proteinase k E.Coli DNA EXTRACTION LYSE THE CELL Tris-HCl AND EDTA Denature the struct-ure of protei-n Remo-ve out protein

6. CONT…. After incubation add chloroform and isoamyl alcohol instead of phenol. Three layers are formed: AQUOS LAYER INTERMEDIATE LAYER BOTTOM LAYER having having having RNA AND DNA DENATURED PROTEIN CHLOROFORM AND ISOAMYL ALCOHOL

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. centrifuge 1.5ml E.coli culture supernatant pellet 600l lysis buffer Vortex and incubate at 37 degree centigrade for 1 hr. Add chloroform and isoamyl alcohol PELLET STEPS OF EXTRACTION OF DNA FROM A E.coli CELL

9. Take aq.layer and centrifuge take supernatant Add 2.5 µ l ethanol Incubate at -20 degree cent. For 30 min. centrifuge

10. Take pellet and add 1ml 70% ethanol centrifuge Take pellet and air dry it Add TE Buffer and incubate

11. ELECTROFORESIS OF ISOLATED DNA

12. 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. INTRODUCTION

13. AGAROSE GEL-: 1% of 100ml is used. means .5g agarose in 49ml d.w and 1ml 50x buffer. 5 µ l EtBr is used. 600ml BUFFER-: (Tank Buffer) 12ml 50x buffer + 588ml d.w. COMPOSITION OF AGAROSE GEL AND BUFFER USED

14. Prepared agarose gel at 45 ºC 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. PROCESS OF GEL ELECTROPHORESIS

15. 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. ROLE OF EtBr

16. RESULT OF ELECTROPHORESIS OF NUCLEIC ACID

17. POLYMERASE CHAIN REACTION OF EXTRACTED DNA FROM E.coli

18. 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. POLYMERASE CHAIN REACTION

19. 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 REQUIREMENT FOR PCR REACTION :

20. 1 step 2 step 3 step 4step Initial denatur annea extent final final Denaturation -ation -ling -ion extent hold TO AMPLIFY 1Kb FRAGMENT FROM EXTRACTED TEMPLATE DNA FROM E.coli 94ºC 2 min. 94 º C 30 sec. 60 º C 30 sec. 72 º C 1 min. 72 º C 2 min. 4 º C 60 min. 32 cycles

21. GRAPH SHOWING PCR REACTION BETWEEN TIME AND TEMPRETURE TEMPRETURE TIME 94 94 2min 30 sec 60 30sec 72 1min 72 2min 4 60min . in ºC

22. REACTION INVOLVED IN PCR . DENATURATION 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ ANNEALING 3’ 5’ 3’ 5’ 5’ 5’ EXTENTION 1 st CYCLE 94 ºC 60ºC 72 ºC

23. . 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ DENATURATION 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ ANNEALING 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 2 nd CYCLE

24. . EXTENTION 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ FORMATION OF 8 DNA STRANDS

25. RESULT OF PCR AFTER ELECTROFORESIS Resulted DNA band of 1kb

26. SDS PAGE

27. 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. PRINCIPLE OF SDS PAGE

28. 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. CONT….

29. STACKING GEL AND RESOLVING GEL : CHEMICALS CONCENTRATION Stacking gel Resolving gel 1. Acrylamide/ .5ml 1.98ml Bisacrylamide 2. Gel buffer 0.63ml 1.25ml (tris-HCl-:1.5M) (pH-:6.8) (pH-:8.8) 3. d.water 1.32ml 1.77ml 4. SDS (10%) 25 µ l 50 µ l 5. TEMED 1 µ l 4 µ l 6. APS (20%) 25 µ l 38 µ l SAMPLE PREPARATION

30. CONT….. ELECTRODE BUFFER : Tris-HCl Glysin SDS 3gm 14.4gm 1gm SAMPLE : 30 µ l protein sample + 30 µ l loading dye. MARKER : 10 µ l DYES : Staining dye distaining dye .8gm coomassie blue d.w-:630ml in 1l d.w glacial acid-:70ml methanol-:300ml

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 sample.it is non-ionic and non-reactive toward proteins to interfering with electricity.

32. Cont.….. Components of LGB and UGB buffer used:- Acrylamide -:When dissolved in water autopolymerization of acrylamide takes place. Joining of molecules head to tail fashion to form single chain polymer. Bisacrylamide- :Cross linking agent for polyacrylamide gel. Two acrylamide molecule coupled head to tail at their non-reactive ends. Hence cross linked two polyacrylamide chains to one another results to a gel formation. TEMED (Tetramethylethylene diamine)-:provide free radicals. APS (Ammonium per sulfate)-:stabilize free radicals and forms the gel.

33. Cont.…… Components of electrode buffer:- 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. SDS -:It bound to the protein.(1.4gm SDS bound 1gm protein)and form SDS bound protein complex. Coats protein with uniform negative charge. Glycine -:Weak acid which is neutral or protonated in the stacking gel while it becomes glycinate or deprotonated in the resolving gel.

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 Cl - but ahead to proteins.

35. Cont.……… Staining dye:- It is anionic, non-polar, non-specifically 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. 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. t o different size.

37. PROCESS OF SDS-PAGE Prepared resolving gel was poured between two glass plates. A fter 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. 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. 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. Cont.…….. For destaining the excessive dye, shaking is provided overnight with the help of decoloring shaker.

41. RESULTS OF SDS PAGE

42. RESTRICTION ENDONUCLEASE

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.

44. CHEMICALS USED IN THE PROCESS CHEMICALS CONCENTRATION IN VIAL 1 CONCENTRATION IN VIAL 2 2X ASSAY BUFFER 12.5 µl 12.5 µl DNA 10 µl 10 µl Eco. R1 1.5 µl _ Hind III _ 1.5 µl

45. PROCEDURE Add these chemicals in two different vials. Then we started electrophoresis. In four different wells, we add In 1 st well DNA digested with EcoR1(vial1) In 2 nd well DNA digested with Hind III(vial2) In 3 rd well standard DNA (undigested DNA) In 4 th well digested DNA

46. RESULTS

47. NANOPARTICLES FROM BIORESOURCES

48. INTRODUCTION OF NANOPARTICLES Less than a nanometer Individual atoms are up to a few tenths of a nanometer in diameter Nanometer Ten shoulder- to-shoulder hydrogen atoms (blue balls) span 1 nanometer. DNA molecules are about 2.5nm wide Thousands of nanometers Biological cells, like these red blood cells, have diameters in the range of thousands of nanometers A million nanometers An ant  is millions of nanometers across Billions of nanometers A two meter tall male is two billion nanometers tall   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.

49. PROCEDURE OF EXTRACTION 0.01gm AgNO 3 dissolved in 100ml distilled water . AgNO 3 dissociate after dissolving in water. AgNO 3 Ag + + NO 3 - can be stabilized by adding bio resource (having large photo -synthetic activity)

50. Cont…….. Leaves of Moringa is taken as bioresource. Leaves were crushed and then centrifuge. Take supernatant . Add supernatant in .1gm AgNO 3 in 100ml distilled water. Kept onto the magnetic stirrer for overnight. Nanoparticles formed by changing the colour of solution.

51. Cont……… . AFTER 2 HOURS AFTER OVERNIGHT STIRRING

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 20 µl,40µl,60µl, 80 µl of nanoparticles were poured and ketoconazole was added in middle well in PDA plate and streptomycin in NA plates. Overnight incubation was given.

53. RESULTS SHOWING ANTIMICROBIAL ACTIVITY OF NANOPARTICLES FOR BACTERIA :- Species concentration of diameter of nanoparticles inhibition zon e E.coli 20 µl 9mm 40 µl 10mm 60 µl 11mm 80 µl 13mm

54. Cont.………. pseudomonas 20 µl 9mm 40 µl 10.5mm 60 µl 11mm 80 µl 12.5mm FOR FUNGI : Sps. Conc. of diameter of zone nanoparticles of inhibition A.niger 20 µl 7mm 40 µl 9.5mm 60 µl 10mm 80 µl 11mm

55. RESULTS Petriplates Showing Antimicrobial Activity :- Presence of clear zone shows the inhibition of E.co li by silver nanoparticles

56. Cont…….. Presence of clear zone shows inhibition of pseudomonas by nanoparticles

57. Cont………. . Presence of clear zone around the wells indicates the inhibition of fungi ( Aspergilus niger )

58. Dot – ELISA ENZYME LINKED IMMUNOSORBENT ASSAY

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.

60. . DIAGRAM SHOWING THE PROCEDURE OF ELISA

61. PROCEDURE 1 st . 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 strip(Ab) a s specific + a ntigen is absent sample i n sample ( A g)

62. Cont.………. 2 nd . Add enzyme antibody conjugate(antibody-HRP) INCUBATE FOR 20 MIN. , WASH sequence A sequence B No binding with Ab-HRP+ enzyme linked ( Ag+strip(Ab) ) antibody

63. Cont.……………. 3 rd . 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, 2H 2 O 2 2H 2 O + O 2 HRP + O 2 BLUE COLOUR TMB

64. PLANT TISSUE CULTURE

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. 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% HgCl 2 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.

67. RESULTS OF PLANT TISSUE CULTURE

68. Cultivation of Spirulina Platensis by using of Zarrok's media

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. Composition of Zarrok’s Media NaHCo 3 - 4.8gm/lt NaNo 3 - 2.5gm/lt NaCl - 1gm/lt K 2 So 4 - 1gm/lt K 2 HPo 4 - 0.5gm/lt MgSo 4 .7H 2 O- 0.2gm/lt FeSo 4 .7H 2 O- 0.01gm/lt CaCl 2. 2H 2 O - 0.04gm/lt EDTA- 0.08gm/lt

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. RESULTS After 9 days incubation culture was at harvesting stage.