Analiza enzymów izolacyjnych i restrykcyjnych DNA
At the end of this lab, students should know...
Q1: What materials do you need to prepare before starting a DNA isolation lab?
Gather popsicle sticks, funnels, 15 mL conical tubes, micropipettes, ice buckets with ice, small cups, and microcentrifuge tubes. Prepare two water baths set to 56°C and 37°C for different lab stages. Limit the lab to no more than 12 samples per class to ensure smooth execution and proper handling of all materials.
Q2: How do you prepare TE buffer for DNA isolation?
Measure 400 mL of reverse osmosis (RO) water, then add 5 mL of 1M Tris-HCl pH 8 and 1 mL of 0.5 M EDTA solution. Fill the remaining volume to 500 mL with RO water. This buffer protects DNA during isolation and restriction enzyme analysis procedures.
Q3: What is the difference between TE buffer and TES buffer?
TES buffer includes all TE buffer components—400 mL RO water, 5 mL of 1M Tris-HCl pH 8, and 1 mL of 0.5 M EDTA—plus an additional 5 mL of 10% SDS solution. The SDS detergent helps lyse cell membranes during DNA extraction. Both are brought to 500 mL final volume with RO water.
Q4: How should you prepare the saline solution for cheek cell collection?
Dissolve 5 grams of NaCl in 500 mL of reverse osmosis water in a clean soda bottle to create a 1% saline solution. Distribute 10 mL aliquots into small drinking cups, providing one cup per student group for collecting cheek cells during the lab.
Q5: Why is ethanol stored in the freezer or refrigerator before the lab?
Ethanol is stored ice-cold to facilitate DNA precipitation during the isolation process. Prepare 10 mL aliquots of 100% ethanol and keep them refrigerated or frozen. Cold ethanol increases DNA solubility differences, making it precipitate more effectively from the aqueous solution.
Q6: What temperature settings are required for the water baths in this lab?
Set one water bath to 56°C for part one of the laboratory exercise and a second water bath to 37°C for part two. These specific temperatures optimize enzyme activity and cell lysis during different stages of DNA isolation and restriction enzyme analysis.
Q7: How much TE buffer or TES buffer should you prepare for a typical lab class?
Prepare 500 mL of TE buffer or TES buffer as the standard preparation volume. Since the lab accommodates up to 12 samples per class, this volume provides sufficient buffer for cell lysis, DNA extraction, and subsequent washing steps across all student groups.