MMGL- Life sciences

Ordering Information
Price: 400/- PKR
Edition: 2023-2024 (2nd edition)
ISBN: 978-627-7502-05-8 (Print)
Published by Dr. Muhammad Ali

Ninety-five (95) full-length experiments are included

© Copyright 2023. All rights reserved by Dr. Muhammad Ali

Contact us for purchase and online ordering:
[email protected]

Video lectures of almost all the experiments will be uploaded sequentially.

MMGL- Life sciences
MMGL- Life sciences
Life Sciences by Dr. Muhammad Ali

Contents of EBB Series-II (Green)

  1. Molecular Biology. 1

Experiment 1.1- Total Genomic DNA extraction from plant by the CTAB method. 5

Experiment 1.2- DNA extraction from fungi 6

Experiment 1.3- Miniprep extraction of plasmid from bacteria. 6

Experiment 1.4- Total genomic DNA extraction from mouse tail/tissue. 7

Experiment 1.5- Quick tail DNA extraction (Jackson’s lab) 8

Experiment 1.6- DNA extraction from blood. 8

Experiment 1.7- Quantification of DNA and RNA by nanodrop. 9

Experiment 1.8- Designing of primers for endpoint PCR from coding sequence of EI24. 10

Experiment 1.9- Designing of primers for qPCR to check transcription of EI24. 15

Experiment 1.9- Polymerase chain reaction (PCR) for genotyping. 20

Experiment 1.10- Agarose gel electrophoresis of PCR product ~150 bp long. 21

Experiment 1.11- Restricting fragment length polymorphism (RFLP) 22

Experiment 1.12- Quality test of RNA by agarose gel electrophoresis. 22

Experiment 1.13- Polyacrylamide gel electrophoresis (PAGE) of PCR product. 22

Experiment 1.14- Gel extraction protocol (Qiagen Gel Extraction Kit) 24

Experiment 1.15- Purification of PCR product and enzyme-mediated digestion. 24

Experiment 1.16- RNA extraction from cultured cells/tissue. 25

Experiment 1.17- Reverse transcriptase polymerase chain (RT-PCR) reaction. 25

Experiment 1.18- Quantitative PCR (qPCR) and operation of the software. 26

Experiment 1.19- Tissue sample lysis for Western blot analysis. 28

Experiment 1.20- Lysis of cultured cells for Western blot analysis. 28

Experiment 1.21- Ammonium sulfate-facilitated protein concentration. 29

Experiment 1.23- Protein quantification by BCA method and protein normalization. 30

Experiment 1.24- SDS-PAGE gel for protein samples. 31

Experiment 1.25- Continuous gradient SDS-PAGE for protein samples. 32

Experiment 1.26- Coomassie Brilliant Blue (CBB) staining for SDS-PAGE. 33

Experiment 1.27- Silver staining for SDS-PAGE gel 33

Experiment 1.28- Western blotting of protein samples obtained from cells and tissues. 34

Experiment 1.29- Protein immunoprecipitation to find protein-protein interaction. 36

  1.  Basic Tools of Bioinformatics 38

Experiment 2.1- Use NCBI to retrieve sequence of PGC1 alfa gene, mRNA, and protein. 39

Experiment 2.2- Use NCBI to retrieve coding and protein sequence of EI24 gene. 42

Experiment 2.3- Translate DNA sequence into RNA sequence. 42

Experiment 2.4- Aligning nucleotide and amino acid sequence of EI24 from human and mouse. 42

Experiment 2.5- How you will draw dandogram among different species taking sequence of TERT. 42

Experiment 2.6- Drawing dandogram by using data obtained from RAPD primers. 43

Experiment 2.7- How will you retrieve data and draw phylogenetic tree. 43

Experiment 2.8- Analysis of physiochemical parameters of proteins. (M. Weight, pI, instability index) 45

Experiment 2.9- Determining of the hydrophobicity or hydrophilicity of protein. 45

Experiment 2.10- Prediction of trans-membrane helix. 46

Experiment 2.11- Secondary structure prediction. 46

Experiment 2.12- Structure Prediction. 46

Experiment 2.13- Codon optimization. 46

Experiment 2.14- Designing gene specific primers using primer blast, primer 3, and primer quest  47

Experiment 2.15- How you will design qPCR primers (SYBR green and probe based) for EI24 gene. 47

Experiment 2.16- Authentication of primers using online tools (oligo analyzer and ). 47

Experiment 2.17- Finding structure (secondary and tertiary) of EI24 proteins using online tools. 48

Experiment 2.18- How you will evaluate 3D structure of EI24 protein. 48

Experiment 2.19- How you will perform gene set enrichment analysis. 48

Experiment 2.20- How will you find signature genes for autophagy and breast cancer. 48

Experiment 2.21- Finding correlation of EI24 with signature genes of autophagy and breast cancer. 49

Experiment 2.22- Oncomine data to find effect of mRNA level of you gene of interest on a specific cancer type. 49

Experiment 2.23- Prediction of Drug-Receptor interaction Using MOE. 49

  1. Cell Biology. 51

Experiment 3.1- Study of cheek cells using methylene blue staining. 54

Experiment 3.2- Study of onion cells and using iodine staining. 54

Experiment 3.3- Observation of fungi cells by lactophenol cotton blue staining. 55

Experiment 3.4- Differentiation between gram +ve and gram –ve bacteria. 55

Experiment 3.5- Stamen hair cells of Tradescantia (light microscopy) 57

Experiment 3.6- Studying different stages of mitosis in onion root tip cells. 57

Experiment 3.7- Study of different stages of meiosis in Tradescantia ohiensis. 58

Experiment 3.8- Different stages of meiosis in the testes of grasshopper. 58

Experiment 3.9- Observing chromosome obtained from blood cells. 59

Experiment 3.10- Observing chromosome obtained from adherent cells (HeLa) 59

Experiment 3.11- Immunohistochemistry (IHC) using anti BrdU antibody. 60

Experiment 3.12- Histology cell shape of skeletal, cardiac, and smooth muscles. 62

Experiment 3.13- White adipose tissue. 63

Experiment 3.14- Liver anatomy and histology. 63

Experiment 3.15- Difference of seed and stem of monocots and dicots. 64

Experiment 3.16- Subculturing adherent mammalian cells. 65

Experiment 3.17- Reviving mammalian cell lines. 66

Experiment 3.18- Cell counting for mammalian cell lines. 66

Experiment 3.19- Cell freezing protocol from ATCC. 67

Experiment 3.20- Thawing of suspension cells. 68

Experiment 3.21- Transfection using lipofectamine. 68

Experiment 3.22- Co-transfection of plasmid DNA and siRNA.. 69

Experiment 3.23- Electroporation of U2OS cell line. 69

Experiment 3.24- Lentivirus production to knockdown TERT mRNA.. 70

Experiment 3.25- Lentivirus Infection and knockdown of TERT mRNA.. 71

Experiment 3.26- Stable cell line expressing EI24 (Transfection method) 72

Experiment 3.27- Preparation of Mouse Embryonic Fibroblasts (MEFs) 73

Experiment 3.28- Luciferase reporter assay. 74

Experiment 3.29- Immunocytochemistry (ICC) for the detection of two proteins. 74

Experiment 3.30- Fluorescently Activated Cell Sorting (FACS) analysis. 75

Experiment 3.31- Determination of cell cycle by FACS. 77

Experiment 3.32- MTT assay for cell viability. 78

Experiment 3.33- Colonogenic assay (crystal violet assay) 78

  1. Recombinant DNA Technology and Genetic Engineering. 80

Experiment 4.1- Preparation of competent cell 81

Experiment 4.2- Transformation of plasmid containing mutated sequence of TRAF6. 82

Experiment 4.3- TA cloning and selection by blue & white colonies. 83

Experiment 4.4- PCR-facilitated introduction of restriction sites and digestion. 84

Experiment 4.5- Restriction of MMTV- ERT2CreERT2 and gel elution. 85

Experiment 4.6- Ligation of the insert into the vector. 86

Experiment 4.7- PCR-facilitated deletion of RING domain from human TRAF6. 87

Experiment 4.8- PCR-facilitated generation of the point mutation (Pro to Leu) 88

Experiment 4.9- PCR-facilitated addition of HA tag at N-term of TRAF6. 90

Experiment 4.10- Production of recombinant insulin. 92

Experiment 4.11- CRISPR/Cas9 for the generation of Tert knockout cell lines and mice. 94

Experiment 4.12- CRISPR/Cas9 to generate Tert knock-in mice at Rosa26 locus. 96

Experiment 4.13- Analyzing mutations generated in Tert knockout cell line/ mice. 98

  1. Appendices. 100

4.1 Cell lines and media. 100

4.2         Useful numbers for cell culture. 100

4.3 Some restriction enzymes. 101

4.4 Reagents for Biochemistry and Biotechnology lab. 102

4.5- Composition for Tris-glycine SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 103

  1. Bibliography and further reading. 104

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