Brief Summary
This video provides a step-by-step guide on how to perform a restriction digest of Lambda DNA using the restriction enzymes PstI, EcoRI, and HindIII. It covers the necessary materials, proper labeling and handling of samples, precise liquid transfers, and incubation procedures. The final step involves preparing the digested samples for gel electrophoresis analysis.
- Use color-coded tubes for easy identification.
- Ensure accurate pipetting techniques for precise measurements.
- Properly mix and incubate samples for optimal digestion.
Introduction
The video introduces the process of performing a restriction digest of Lambda DNA using PstI, EcoRI, and HindIII restriction enzymes. This activity involves digesting DNA from bacteriophage Lambda with specific restriction enzymes.
Materials Needed
To perform the restriction digest, you'll need EcoRI, HindIII, and PstI restriction enzymes in microcentrifuge tubes on ice. Additionally, you'll require samples of Lambda DNA and 2x restriction buffer, also kept on ice. Color-coded tubes are recommended for organization.
Preparing the Samples
Label four color-coded microcentrifuge tubes with "L" for Lambda DNA, "E" for EcoRI, "P" for PstI, and "H" for HindIII. Include your initials and the date on each tube. Place the tubes in a microcentrifuge tube rack to keep them organized during the experiment.
Adding Lambda DNA
Using a fresh pipette tip for each tube, transfer 4 microliters of Lambda DNA from the stock tube to the corresponding color-coded tubes. Ensure the pipette tip is properly submerged in the sample and that the correct amount is drawn up. Dispense the sample into the matching tube, placing the tip near the bottom to ensure complete transfer.
Adding 2x Restriction Buffer
Reset the micropipette and use a new tip to transfer 6 microliters of 2x restriction buffer to the tube labeled "L". For the tubes labeled "E," "P," and "H," transfer 5 microliters of 2x restriction buffer to each, using a fresh tip for each transfer to avoid contamination.
Adding Restriction Enzymes
After adding the restriction buffer, reset the micropipette and use a fresh tip to transfer 1 microliter of the appropriate restriction enzyme to its corresponding tube. This ensures that each enzyme is added to the correct Lambda DNA sample.
Mixing and Centrifuging
Mix the components in each tube by gently flicking them to ensure thorough mixing. Then, place the tubes in a microcentrifuge, ensuring the load is properly balanced. Spin the samples briefly to collect the liquid at the bottom of each tube.
Incubation
After centrifuging, place the tubes in a rack and incubate them for 30 minutes in an incubator or water bath at 37°C, or overnight at room temperature. This incubation period allows the restriction enzymes to digest the Lambda DNA.
Post-Digestion Analysis
Once the digest reactions are complete, the samples are ready for analysis. The video refers viewers to another video on loading and running an agarose gel to visualize the digested DNA fragments.
