Molecular Biology Techniques by Carson, Sue; Miller, Heather B.; Srougi, Melissa C.; Witherow, D. Scott;

A Classroom Laboratory Manual

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Kiadás sorszáma 4
Kiadó Elsevier Science
Megjelenés dátuma 2019. március 11.

ISBN 9780128180242
Kötéstípus Puhakötés
Terjedelem294 oldal
Méret 276x216 mm
Súly 860 g
Nyelv angol

Kategóriák

Sejttan Molekuláris biológia

Hosszú leírás:

Molecular Biology Techniques: A Classroom Laboratory Manual, Fourth Edition is a must-have collection of methods and procedures on how to create a single, continuous, comprehensive project that teaches students basic molecular techniques. It is an indispensable tool for introducing advanced undergraduates and beginning graduate students to the techniques of recombinant DNA technology-or gene cloning and expression. The techniques used in basic research and biotechnology laboratories are covered in detail. Students will gain hands-on experience on subcloning a gene into an expression vector straight through to the purification of the recombinant protein.

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Tartalomjegyzék:

Part I: Manipulation of DNAAdvanced Alternatives Within Part I 1. Getting Oriented; Practicing With Micropipettes 2. Purification and Digestion of Plasmid (Vector) DNA3. Completion of Vector Preparation and Polymerase Chain Reaction Amplification of egfp 4. Preparation of Insert DNA (egfp) PCR Product5. DNA Ligation and Transformation of Escherichia coli

Part II: Screening TransformantsAdvanced Alternatives Within Part II6. Screening of Transformants, Part I 7. Screening of Transformants, Part II8. Analysis of DNA Sequence From a Positive Clone, Part II

Part III: Expression, Detection, and Purification of Recombinant Proteins from BacteriaAdvanced Alternatives Within Part III 9. Expression of Fusion Protein from Positive Clones, SDS_PAGE and Western Blot: Part I 10. Expression of Fusion Protein from Positive Clones, SDS_PAGE, and Western Blot: Part II 11. Extraction of Recombinant Protein From Escherichia coli Using a Glutathione Affinity Column12. Analysis of Purification Fractions

Part IV: Analysis of mRNA Levels13. Total RNA Purification14. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part I15. Analysis of gst::egfp mRNA Levels by RT-qPCR: Part II 16. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part I 17. Analysis of gst::egfp mRNA Levels by Semiquantitative RT-PCR: Part II

Part V: Modulation of Gene Expression18. Culturing Mammalian Cells19. Transient Transfection of Mammalian Cells20. RNAi-Mediated Knockdown of EGFP: Part I 21. RNAi-Mediated Knockdown of EGFP: Part II22. RNAi-Mediated Knockdown of EGFP: Part III23. CRISPR-Mediated Knockout of EGFP: Part I24. CRISPR-Mediated Knockout of EGFP: Part II 25. Advanced CRISPR: Part I 26. Advanced CRISPR: Part II27. Interim Laboratory Session 28. Advanced CRISPR: Part IV 29. Advanced CRISPR: Part V30. Advanced CRISPR: Part VI

AppendixA: Equipment B: Prep List C: Preparation of Competent Escherichia coli Cells D: Pre-Lab Questions