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BT734 : Research Methods in Biotechnology

Course Overview

Course Synopsis

This course is designed to equip students with the multidisciplinary skills and knowledge required in the field of biotechnology. In this course, students will learn the basic skills of the biotechnology workplace, aseptic techniques, preparation of the culture media, isolation of plasmid DNA, quantitation of DNA, polymerase chain reaction, restriction digestion and agarose gel electrophoresis, southern transfer, preparation, purification, and hybridization of probe, transformation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blotting and DNA sequencing. This course will enable the students to get sufficient knowledge about the subject to carry out research and pursue career in the same field.

Course Learning Outcomes

After the successful completion of the course, students will be able to

  • Develop essential research and practical skills in the field of biotechnology.
  • Prepare different types of culture media
  • Isolate and quantify plasmid DNA
  • Perform PCR, restriction digestions, agarose gel electrophoresis, PAGE, Western blotting and DNA sequencing
  • Work independently and take initiative in solving the research problems that may be encountered

Course Calendar

1 Format of Student Laboratory Records
2 Format of Student Laboratory Records 2
3 The Ten Commandments of Record Keeping
4 The Ten Commandments of Record Keeping 2
5 Record Keeping and Safety Rules
6 Record Keeping and Safety Rules 2
7 Record Keeping and Safety Rules 3
8 The Basic Skills of the Biotechnology Workplace: Measuring Volumes
9 The Basic Skills of the Biotechnology Workplace: Measuring Volumes using Pipettes
10 The Basic Skills of the Biotechnology Workplace: Measuring Volumes using MIcropipettes
11 The Basic Skills of the Biotechnology Workplace: Measuring Volumes using MIcropipettes 2
12 Basic Skills of the Biotechnology Workplace: Making Solutions
13 Basic Skills of the Biotechnology Workplace: Solutions: Mass/Volume Concentration
14 Basic Skills of the Biotechnology Workplace: Solutions of Differing % Mass/Volume Concentrations
15 Solutions of differing Molar Concentrations
16 Basic Skills of the Biotechnology Workplace: Solutions; Parallel dilution of Existing Stock solution
17 Basic Skills of the Biotechnology Workplace: Solutions; Serial dilution of Existing Stock solution
18 Aseptic Techniques: Introduction
19 Aseptic Techniques: Purpose
20 Aseptic Techniques: Types

21 Aseptic Technique & Establishing Pure Cultures: Introduction
22 Aseptic Technique & Establishing Pure Cultures: Reagents and Supplies
23 Aseptic Technique & Establishing Pure Cultures: Equipment
24 Aseptic Technique & Establishing Pure Cultures: Streak Plate
25 Aseptic Technique & Establishing Pure Cultures:Culture transfer
26 The Growth Curve: Introduction
27 The Growth Curve: Reagents and Supplies
28 The Growth Curve: Equipment
29 The Growth Curve: Procedure
30 The Growth Curve: Cell count using hemocytometer
31 The Growth Curve: Spectrophotometer
32 The Growth Curve: Spectrophotometer Types and Procedure
33 Preparation of the Culture Media: Introduction
34 Preparation of culture Media: Reagents and Supplies
35 Preparation of culture Media: Equipment
36 Preparation of Culture media: Preparation of YEPD (Complex) medium
37 Preparation of Culture Media: Preparation of YNB (Defined medium)
38 DNA Isolation Methods: Preparation of Total Cell DNA
39 Isolation of Plasmid DNA: The Mini-Prep: Introduction
40 Isolation of Plasmid DNA: The Mini-Prep: Reagents and Supplies

41 Isolation of Plasmid DNA: The Mini-Prep: Equipment
42 Isolation of Plasmid DNA: The Mini-Prep: procedure
43 Purification of DNA: Introduction
44 Purification of Plasmid DNA by solvent extraction: Reagents ,supplies and equipment
45 Purification of Plasmid DNA by solvent extraction: Procedure
46 Concentration of Plasmid DNA: Reagents , supplies and equipment
47 Concentration of Plasmid DNA: Procedure
48 Quantitation of DNA: Reagents ,supplies and equipments
49 Quantitation of DNA: Procedure (UV absorption)
50 Quantitation of DNA: Procedure (Etbr Flourescence)
51 Large Scale Isolation of Plasmid DNA (Column chromatography): Introduction
52 Large Scale Isolation of Plasmid DNA (Column chromatography):Ion Exchange Chromatography
53 Large Scale Isolation of Plasmid DNA (Column chromatography):Reagents\Supplies
54 Large Scale Isolation of Plasmid DNA (Column chromatography): Equipments
55 Large Scale Isolation of Plasmid DNA (Column chromatography): Procedure
56 Large Scale Isolation of Plasmid DNA (Column chromatography):CsCl Density gradient
57 Amplification of a gene by (PCR): Introduction
58 Amplification of a gene by (PCR): Parameters that affect PCR
59 Amplification of Gene Fragment by PCR: Thermostable DNA Polymerases
60 Amplification of Gene Fragment by PCR: Programing a PCR

61 Amplification of Gene Fragment by PCR: Selecting PCR Primers
62 Amplification of Gene Fragment by PCR: Properties of Oligonucleotides and efficiency of amplification
63 Amplification of Gene Fragment by PCR: Calculating melting temperature ™
64 Amplification of Gene Fragment by PCR: Contamination in PCR
65 Amplification of Gene Fragment by PCR: Reagents and Supplies
66 Amplification of Gene Fragment by PCR: Procedure
67 Amplification of Gene Fragment by PCR: Trouble shooting the amplification
68 Amplification of Gene Fragment by PCR: Troule shooting major problems that occur in PCR
69 Amplification of Gene Fragment by PCR: Purification of PCR Products in Preparation for Cloning
70 Purification of PCR products: Reagents, Supplies and Equipment
71 Purification of PCR products: Procedure
72 Removal of Primers and Excess dNTPs from Amplified DNA by Ultrafication: Introduction
73 Removal of Primers and Excess dNTPs from Amplified DNA by Ultrafication: Reagents supplies and Equipment
74 Removal of Primers and Excess dNTPs from Amplified DNA by Ultrafication: Procedure
75 Amplification of Gene fragment by PCR: Types of PCR
76 Gel Electrophoresis: Introduction
77 Agarose Gel electrophoresis: Agarose
78 Agarose Gel electrophoresis: The rate of migration of DNA through agarose gels
79 Agarose Gel electrophoresis: Electrophoresis Buffers
80 Gel electrophoresis: Gel Loading Buffer

81 Agarose Gel electrophoresis: Reagents ,Supplies and Equipments
82 Agarose Gel electrophoresis: Procedure
83 Agarose Gel Electrophoresis: Detection of DNA in Agarose gel
84 Agarose Gel Electrophoresis: Staining with Sybr Gold
85 Agarose Gel Electrophoresis: Photography of DNA in Gel
86 Agarose Gel Electrophoresis: Recovery of DNA from Low-melting-temperature Agarose Gels Organic Extraction
87 Agarose Gel Electrophoresis: Reagents and supplies Recovery of DNA from Low-melting-temperature Agarose Gels Organic Extraction
88 Agarose Gel Electrophoresis: Procedure Reagents and supplies Recovery of DNA from Low-melting-temperature Agarose Gels Organic Extraction
89 Southern Hybridization: Introduction
90 Southern Hybridization: Methods to transfer DNA from Agarose gel to solid support
91 Southern Hybridization: Membranes used for Southern and Northern blotting
92 Southern Hybridization: Capillary transfer method Southern Blot
93 Southern Hybridization: Restriction digestion of DNA for southern
94 Southern Hybridization: Restriction digestion [Problems and Solutions]
95 Southern Hybridization: Reagents and Supplies
96 Southern Hybridization:Procedure [Digestion & Electrophoresis]
97 Southern Hybridization: Preparation of gel for transfer
98 Southern Hybridization:Preparation of membrane for transfer
99 southern hybridization:Apparatus assembly for DNA transfer
100 Southern Hybridization:Fixation of DNA to membrane

101 Southern Hybridization: Southern blot: hybridization of radiolabeled probe to nucleic acids
102 Probe hybridization to Nucleic Acids:Reagents and Supplies
103 Probe hybridization to Nucleic Acids:Hybridization solutions for different buffers
104 Probe hybridization to Nucleic Acids:Salmon Sperm DNA
105 Probe hybridization to Nucleic Acids:Special Equipment
106 Probe hybridization to Nucleic Acids:Procedure
107 Probe hybridization to Nucleic Acids: Stripping Probes from Membranes
108 Southern Hybridization:Method: Stripping Probes from Membranes
109 Southern Hybridization:Method: Stripping Probes (Nylon Membrane)
110 Southern Hybridization: Probe synthesis:Nick Translation radioactive labeling

111 Southern Hybridization: Probe synthesis:End filling radioactive labeling
112 Southern Hybridization: Probe synthesis:Random primed radioactive labeling
113 Random Priming; Radiolabeling of DNA:Introduction
114 Random Priming; Radiolabeling of DNA:Reagents and Supplies
115 Random Priming; Radiolabeling of DNA:Procedure
116 Purification of RNA from Cells & Tissues:Introduction
117 Purification of RNA from Cells & Tissues:Reagent & Supplies (Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction
118 Purification of RNA from Cells & Tissues:Procedure
119 Purification of RNA from Cells & Tissues:Procedure_2
120 Purification of RNA from Cells & Tissues:Procedure_3
121 Purification of RNA from Cells & Tissues:Procedure [Extraction of RNA from Homogenates

122 Purification of RNA from Cells & Tissues:Storage of RNA
123 Simultaneous Preparation of DNA, RNA, & Protein:Introduction
124 Simultaneous Preparation of DNA, RNA, & Protein:Reagent & Supplies
125 Simultaneous Preparation of DNA, RNA, & Protein:Procedure
126 Simultaneous Preparation of DNA, RNA, & Protein:Procedure_2
127 Simultaneous Preparation of DNA, RNA, & Protein:Procedure_3
128 Simultaneous Preparation of DNA, RNA, & Protein:Procedure_4
129 Purification of Poly A RNA:Introduction
130 Purification of Poly A RNA:Reagents & Supplies
131 Purification of Poly A RNA:Procedure
132 Purification of Poly A RNA:Further Purification of Eluted RNA
133 Northern Hybridization:Introduction
134 Electrophoresis [Glyoxylated RNA]:Introduction
135 Electrophoresis [Glyoxylated RNA]:Reagents and supplies
136 Electrophoresis [Glyoxylated RNA]:Procedure
137 Quality of RNA:How to check quality of RNA preparation
138 Gel Electrophoresis [Formaldehyde]:Introduction
139 Gel Electrophoresis [Formaldehyde]:Reagents and supplies
140 Gel Electrophoresis [Formaldehyde]:Procedure

141 Transfer and Fixation of Denatured RNA to membrane Introduction (1)
142 Reagents and Supplies for transfer and fixation of denatured RNA
143 Procedure Transfer and Fixation of denatured RNA
144 Procedure Assembly of the transfer system
145 Procedure Staining of RNA and fixation of RNA on membrane
146 Northern Blotting introduction
147 Reagents and Supplies for Northern blotting
148 Northern blotting: Procedure
149 Protein assay Introduction Biuret
150 Reagents and Supplies for Biuret
151 Procedure biuret assay
152 Protein assay Introduction Lowry
153 Reagents and Supplies for Lowry assay
154 Procedure Lowry assay
155 Bradford protein assay
156 Reagents and Supplies Bradford protein assay
157 Procedure Bradford assay
158 Protein Purification from Inclusion bodies: Introduction
159 Protein Purification from Inclusion bodies: Reagent and Supplies
160 Purification of Proteins: Procedure (Preparation of cell Extract)

161 Purification of Proteins: Procedure (Purification and washing of inclusion bodies)
162 Purification of Proteins: Procedure (Purification and washing of inclusion bodies) 1
163 Purification of Proteins: Procedure (Solubilization of Inclusion Bodies)
164 Protein Isolation from Animal Tissues: Introduction
165 Protein Purification from Animal Tissues: Reagents and Supplies
166 Protein Extraction from Animal Tissues: Procedure
167 Precipitation with Ammonium Sulfate: Introduction
168 Precipitation with Ammonium sulfate:Reagents & Supplies
169 Precipitation with Ammonium sulfate:Procedure
170 SDS-PAGE:Introduction
171 SDS-PAGE:Reagents & Supplies
172 SDS-PAGE:Reagents & Supplies 2
173 SDS-PAGE:Procedure
174 SDS-PAGE:Procedure 2
175 SDS-PAGE:Procedure Staining
176 Western Blotting:Introduction
177 Transfer of protein to membrane:Procedure
178 Transfer of protein to membrane:Procedure2
179 PROBE FOR WESTERN BLOT:Reagents & Supplies
180 Immunoblotting:Procedure

181 ELISA:Introduction
182 ELISA:Types of ELISA
183 ELISA:Sandwich ELISA
184 ELISA:COMPETITION OR INHIBITION ELISA
185 ELISA:Reagents & Supplies
186 ELISA:Procedure
187 ELISA RESULTS:Results
188 Microarray:Introduction
189 MICROARRAY:Types of Microarray
190 Microarray:Reagents & Supplies
191 Microarray:Procedure
192 Transgenic Mouse (TM) Preparation:Introduction
193 Transgenic Mouse (TM) Preparation:Reagents & Supplies
194 Transgenic Mouse (TM) Preparation:Animals and Drugs
195 Transgenic Mouse (TM) Preparation:Procedure Egg collection
196 Transgenic Mouse (TM) Preparation:Procedure Egg collection 2
197 Transgenic Mouse (TM) Preparation:Procedure;Injection of eggs
198 Transgenic Mouse (TM) Preparation:Procedure;Implanting Eggs in Foster Mothers
199 DNA Sequencing:Introduction
200 DNA Sequencing:Types of DNA Sequencing

201 DNA Sequencing:The Chemical Method of DNA Sequencing
202 Preparing template for ddNTP Sequencing:Introduction
203 Preparing template for ddNTP Sequencing:Reagents & Supplies
204 Preparing template for ddNTP Sequencing:Procedure
205 ddNTP Sequencing; Taq DNA Polymerase:Introduction
206 ddNTP Sequencing; Taq DNA Polymerase:Reagents & Supplies
207 Preparing template for ddNTP Sequencing:Procedure 2
208 ddNTP sequencing using Sequenase:Introduction
209 ddNTP Sequencing; Sequenase:Reagents & Supplies
210 Preparing template for ddNTP Sequencing:Procedure(Sequenase)
211 ddNTP sequencing; Klenow fragment:Introduction
212 ddNTP Sequencing; Taq DNA Polymerase:Reagents & Supplies 2
213 ddNTP Sequencing; Klenow:Procedure
214 ddNTP (Cycle) Sequencing:Introduction(Using PCRand End- labeled Primers)
215 ddNTP (Cycle) Sequencing:Introduction;Advantage
216 ddNTP (Cycle) Sequencing:Reagents & Supplies
217 ddNTP Cycle Sequencing:Procedure
218 Chemical Sequencing:Introduction
219 Chemical Sequencing:Reagents & Supplies
220 Chemical Sequencing:Procedure

221 Cell Culture:Introduction
222 Cell Lines:Introduction Cell Lines
223 Cell Culture types:Introduction
224 When to Subculture:Introduction
225 Cell Culture:Equipment
226 Cell Culture:Laboratory
227 Cell Culture:Cell culture Hood Layout
228 Cell Culture:Laboratory III
229 Cell Culture:Laboratory IV
230 Trypsinizing and Subculturing:Trypsinizing and subculturing cells from a monolayer
231 Trypsinizing and Subculturing:Procedure:Trypsinizing and subculturing cells from a monolayer

232 Cell Passage:Passaging cells in Suspension Culture
233 Freezing Human Cell:Freezing Human cells grown in monolayer cultures
234 Suspension Culture:FREEZING CELLS GROWN IN SUSPENSION CULTURE
235 Thaw and Recovery:Thawing and recovering human cells
236 Thaw and Recovery:Procedure
237 HEMOCYTOMETER AND TRYPAN BLUE STAINING:DETERMINING CELL NUMBER AND VIABILITY
238 Cell Transport:PREPARING CELLS FOR TRANSPORT
239 siRNA [Small interfering RNA]:Introduction
240 siRNA: Where do they come from?
241 siRNA:siRNA designing
242 siRNA:Mechanism
243 siRNA:Applications

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