Keywords
LB/SOB/SOC media (autoclave temps, antibiotic use), Buffer recipes (Tris-HCl, EDTA, NaCl; pH & molarity), Alkaline lysis miniprep (P1/P2/N3; RNase A), A260/280 & A260/230 (1 A260 = 50 µg/mL dsDNA), Beer–Lambert (c = A/εl; pathlength corrections), Restriction digestion setup (units, % glycerol ≤10, star activity), Sticky vs blunt ends (compatible overhangs),T4 DNA ligase (molar ratios 3:1 insert:vector; PEG effect), Agarose % vs fragment size (0.8–2.0%; TAE vs TBE), DNA stains (EtBr/SYBR; safety & disposal), Competent cells (CaCl₂) (ice-cold prep; OD₆₀₀ ≈ 0.4–0.6),Heat-shock / Electroporation (42 °C 45–60 s / 1.8 kV cuvette), Recovery media (SOC 30–60 min; antibiotic lag), Transformation efficiency = colonies × dilution ÷ µg DNA (CFU/µg), Blue–white screening (lacZ, X-gal/IPTG; α-complementation), Selectable markers (Ampᴿ/Kanᴿ; working concentrations), Colony PCR (template prep, primer design across MCS),Diagnostic digest (single/double; expected banding), Gel documentation (ladder choice; log-size plotting), Record-keeping & biosafety (labels, MSDS, waste segregation), ICAR SyllabusMethods in Recombinant DNA Technology: Volume 11 : The Question Bank Series - Volume 11
authored by: Archana Rani & Yogendra SinghLength: 152 mm | Breadth: 16.5 mm | Height: 229 mm | Imprint: CONTENT VIBES | Weight: 580 GMS
This book will be available from 10-Jan-2026
This lab-focused manual converts your 10 practicals into step-by-step SOPs with checklists, calculations, controls, and troubleshooting—exactly what exams and vivas look for. Each exercise (growth media → buffers → miniprep → QC → digestion → gel → competent cells → E. coli transformation → screening → confirmation) includes:
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Materials & prep tables (reagent recipes, stock→working dilutions, pH setpoints).
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Critical parameters (temperatures, times, rpm, enzyme units, percent agarose).
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Controls (positive/negative, vector-only, undigested DNA).
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Result criteria (acceptance ranges: A260/280, band sizes, colony patterns).
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Viva boxes (why steps work: alkaline lysis chemistry, star activity, blue-white logic).
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Troubleshooting maps (no colonies, smeared gels, low DNA yield, non-specific PCR).
Mini-numericals appear where needed: A260 quant, insert:vector molar ratio, digestion setup, spray-and-plate volumes, and transformation efficiency.
Archana Rani: Senior Research Fellow, Department of Plant Breeding & Genetics, College of Agriculture, Jawahar Lal Nehru Krishi Vishwavidyala, Jabalpur, Madhya Pradesh
Yogendra Singh: Senior Assistant Professor (Biotechnology),Department of Genetics and Plant Breeding, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur-482004, Madhya Pradesh
1 Preparation of Growth, Madia
2 Preparation of Stock Solutions and Buffers
3 Plasmid DNA Isolation
4 Quality and Quantity Assessment of DNA
5 Restriction Digestion of DNA
6 Agarose Gel Electrophoresis
7 Preparation of Competent Cells
8 Preparation of Genetic Transformation of E. Coli
9 Screening of Recombinant DNA Clones in E. Coli.
10 Confirmation of Recombinant Clones