Experiment 01Recombinant DNARestriction Mapping
Recombinant Plasmid Vector Restriction Digestion, Mapping & Agarose Electrophoresis: pUC19 with EcoRI & HindIII
Duration: 4 Hours • Molecular Biology Bench Lab
🎯 Experimental Aim & Learning Objectives
To perform single and double restriction endonuclease digestions of the cloning plasmid vector pUC19 and a recombinant derivative carrying a 1.2 kb foreign cDNA insert using EcoRI and HindIII; resolve digestion fragments on a 1.0% agarose gel alongside DNA molecular weight ladders; and construct an accurate physical restriction map confirming directional insert cloning.
🔬 Theoretical Foundation & Molecular Principles
Restriction endonucleases (Type II) recognize palindromic DNA sequences with extreme specificity, cleaving phosphodiester backbones:
- Vector Architecture (pUC19): A 2686 bp high-copy cloning vector derived from pBR322:
• Multiple Cloning Site (MCS, polylinker): A 54 bp synthetic array embedded in-frame within the 5′ coding region of the lacZα gene, containing unique recognition sites for EcoRI ($5’text{-G}downarrowtext{AATTC-}3’$), SmaI, BamHI, SalI, PstI, and HindIII ($5’text{-A}downarrowtext{AGCTT-}3’$).
• ampR: Encodes $beta$-lactamase conferring ampicillin resistance.
• ori: ColE1/pMB1 origin of replication driving high copy number (~500 copies/cell). - Electrophoretic Migration Forms: Uncut plasmid DNA resolves into distinct topological conformers:
1. Supercoiled (Form I): Compact covalently closed circular (ccc) DNA; migrates fastest.
2. Nicked Circular (Form II): Single-strand nick releases topological tension; migrates slowest.
3. Linearized (Form III): Produced by a single restriction cut; migrates at true molecular size ($2686text{ bp}$). - Double Digestion & Sticky-End Ligation: Digestion with both EcoRI and HindIII generates non-complementary asymmetric overhangs (cohesive 5′ sticky ends), ensuring directional insertion of foreign DNA and preventing vector self-ligation.
Figure 1.1: Physical map of the pUC19 cloning vector ($2686text{ bp}$) highlighting the polylinker (MCS), $amp^R$, and $ori$, alongside agarose gel electrophoresis diagnostic restriction digestion profiles.
🧪 Reagents & Laboratory Equipment
- Enzymes & DNA: Purified pUC19 plasmid DNA ($0.5text{ mu g/mu L}$); recombinant pUC19-insert plasmid; Restriction Endonucleases EcoRI ($10text{ U/mu L}$) and HindIII ($10text{ U/mu L}$); $10timestext{ Buffer rCutSmart}$ (NEB).
- Gel Electrophoresis: Molecular biology grade agarose; $1timestext{ TAE Buffer}$ (40 mM Tris-acetate, 1 mM EDTA, pH 8.3); 1 kb DNA ladder; $6timestext{ TriTrack DNA Loading Dye}$; GelRed nucleic acid stain; horizontal submarine electrophoresis unit with power supply.
📋 Step-by-Step Digestion Protocol
- Reaction Setup: Assemble four $20text{ mu L}$ restriction digests in sterile microcentrifuge tubes:
• Tube 1 (Uncut Control): $2text{ mu L}$ pUC19 DNA + $2text{ mu L } 10timestext{ Buffer} + 16text{ mu L } text{ddH}_2text{O}$.
• Tube 2 (EcoRI Single Digest): $2text{ mu L}$ pUC19 DNA + $2text{ mu L } 10timestext{ Buffer} + 15text{ mu L } text{ddH}_2text{O} + 1text{ mu L EcoRI}$.
• Tube 3 (HindIII Single Digest): $2text{ mu L}$ pUC19 DNA + $2text{ mu L } 10timestext{ Buffer} + 15text{ mu L } text{ddH}_2text{O} + 1text{ mu L HindIII}$.
• Tube 4 (Double Digest Recombinant): $2text{ mu L}$ recombinant DNA + $2text{ mu L } 10timestext{ Buffer} + 14text{ mu L } text{ddH}_2text{O} + 1text{ mu L EcoRI} + 1text{ mu L HindIII}$. - Incubation: Pulse-spin tubes and incubate in a water bath at $37^circtext{C}$ for 60 minutes. Heat-inactivate enzymes at $65^circtext{C}$ for 15 minutes.
- Gel Preparation & Loading: Cast a 1.0% agarose gel in $1timestext{ TAE}$ with $1timestext{ GelRed}$. Add $4text{ mu L } 6timestext{ loading dye}$ to each tube. Load $15text{ mu L}$ of each sample alongside $5text{ mu L}$ of 1 kb ladder.
- Electrophoresis & Imaging: Run at $90text{ V}$ for 75 minutes. Visualize and document bands on a UV/blue-light transilluminator.
📊 Representative Gel Analysis & Sizing Data
| Lane | Sample Contents | Observed Band Sizes | Molecular Interpretation |
|---|---|---|---|
| 1 | 1 kb DNA Ladder | 4.0, 3.0, 2.0, 1.5, 1.0, 0.5 kb | Molecular weight calibration standard |
| 2 | Uncut pUC19 | ~2.0 kb (dominant) & ~3.5 kb (faint) | Form I Supercoiled (runs fast) + Form II Nicked circle |
| 3 | pUC19 + EcoRI | Single crisp band at 2686 bp | Form III Linear vector; authentic molecular size confirmed |
| 4 | Recombinant (EcoRI + HindIII) | 2.68 kb backbone + 1.20 kb insert | Insert successfully released; directional cloning verified |
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