lab homework 1

Miniprep of Plasmid DNA

• Objective: Isolate and quantify plasmid DNA from transformed E. coli for further analysis (e.g., restriction mapping, DNA sequencing).

Overview of Miniprep Procedure

• Transformation & Selection: After transforming E. coli with a plasmid vector containing the desired PCR fragment, colonies are selected on LB + Amp plates.

• Liquid Culture Growth: Individual colonies are picked and grown in liquid culture (LB broth + ampicillin) at 37° C for 12-18 hours to amplify plasmid DNA.

• Isolating Plasmid DNA: Plasmid DNA is extracted from cultures for subsequent analyses to confirm the presence of the desired DNA fragment.

False Clones

• Importance of Verification: Some colonies may be false positives (do not contain the desired insert). These may arise from:

  • Self-ligation of the plasmid.

  • Insertion of extraneous DNA fragments.

• Verification Methods: Use restriction enzyme analysis to confirm the presence of the desired insert in the plasmid DNA.

QIAGEN Plasmid Kits

Procedure Steps

1. Centrifugation: Pellet bacterial cells from liquid culture.

2. Lysis: Use alkaline lysis method:

   • Buffer P1 (with RNase) to resuspend cells.

   • Buffer P2 (SDS + NaOH) to lyse cells.

   • Buffer N3 (acidic potassium acetate) neutralizes lysis.

3. Affinity Purification:

   • Centrifuge to separate plasmid DNA from debris.

   • Utilize wash and elution buffers to isolate clean plasmid DNA.

Binding Principle of QIAGEN Resin

• Mechanism: Negatively charged DNA interacts with positively charged QIAGEN resin during purification.

Buffers Used in Miniprep

Miniprep Procedure Steps

Part I: Observing Transformation Results

• Counting Colonies: Count colonies on transformation plates, record in lab notebook. Plates with >200 colonies should be marked as "TNTC".

• Clonal Populations: Each colony originates from a single E. coli cell.

Part II: Inoculating Cultures (Performed by TA)

1. Add ampicillin for selection of transformed colonies.

2. Inoculate selected colonies into LB+Amp broth.

3. Grow overnight at 37°C with shaking; transfer samples for miniprep.

Part III: Miniprep Procedure

1. Centrifugation: Pellet bacterial cultures and pour off supernatant.

2. Resuspension: Resuspend pellet in Buffer P1.

3. Add Buffer P2: Invert the tube gently to mix and initiate lysis.

4. Add Buffer N3: Neutralize the lysis and form precipitate.

5. Centrifuge: Separate supernatant from precipitate.

6. Column Binding: Transfer supernatant to affinity column; centrifuge and discard flow-through.

7. Wash and Elute: Wash column; add elution buffer to isolate plasmid DNA.

8. Storage: Label and store samples at -20°C.

Quantification of Plasmid DNA

• UV Spectrophotometry: Quantifies nucleic acids by measuring absorbance at specific wavelengths:

  • A260: Absorbance peak indicative of nucleic acid concentration.

  • A260/A280 Ratio: Ideal ratio for pure DNA is 1.8; for RNA, it's 2.0.

  • Outlier ratios can indicate contamination.

NanoDrop Technology

• Functionality: Allows quantification of small sample volumes (0.5 μl - 2.0 μl).

• Usage: Measure purity and concentration of plasmid DNA samples efficiently.

• Cleaning: Wipe pedestal after each measurement to prevent contamination.

Steps for Quantifying Plasmid DNA Samples

1. Bring samples to NanoDrop and follow TA instructions for measurement.

2. Record concentration and purity; perform analyses in duplicates.

3. Select samples based on purity for further experiments.

4. Document needed volumes for restriction analysis and sequencing.