The Alu 1 enzyme comes at a concentration of 5,000 U/ml. Its buffer comes 10X. The DNA is at a concentration of 4 ug/ul. You are ordered to do a digestion reaction of 12 ug of DNA in a total volume of 25 ul. You should use 1 U/ug of DNA. a) In what volume will you get them? b) How will you dispense them? c) Prepare the following table with the reactants of the reaction: Buffer _______ul DNA ______ug _______ul Enzyme ______U _______ul Water _______ul
Molecular Techniques
Molecular techniques are methods employed in molecular biology, genetics, biochemistry, and biophysics to manipulate and analyze nucleic acids (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)), protein, and lipids. Techniques in molecular biology are employed to investigate the molecular basis for biological activity. These techniques are used to analyze cellular properties, structures, and chemical reactions, with a focus on how certain molecules regulate cellular reactions and growth.
DNA Fingerprinting and Gel Electrophoresis
The genetic makeup of living organisms is shown by a technique known as DNA fingerprinting. The difference is the satellite region of DNA is shown by this process. Alex Jeffreys has invented the process of DNA fingerprinting in 1985. Any biological samples such as blood, hair, saliva, semen can be used for DNA fingerprinting. DNA fingerprinting is also known as DNA profiling or molecular fingerprinting.
Molecular Markers
A known DNA sequence or gene sequence is present on a chromosome, and it is associated with a specific trait or character. It is mainly used as a genetic marker of the molecular marker. The first genetic map was done in a fruit fly, using genes as the first marker. In two categories, molecular markers are classified, classical marker and a DNA marker. A molecular marker is also known as a genetic marker.
DNA Sequencing
The most important feature of DNA (deoxyribonucleic acid) molecules are nucleotide sequences and the identification of genes and their activities. This the reason why scientists have been working to determine the sequences of pieces of DNA covered under the genomic field. The primary objective of the Human Genome Project was to determine the nucleotide sequence of the entire human nuclear genome. DNA sequencing selectively eliminates the introns leading to only exome sequencing that allows proteins coding.
The Alu 1 enzyme comes at a concentration of 5,000 U/ml. Its buffer comes 10X. The DNA is at a concentration of 4 ug/ul. You are ordered to do a digestion reaction of 12 ug of DNA in a total volume of 25 ul. You should use 1 U/ug of DNA.
a) In what volume will you get them?
b) How will you dispense them?
c) Prepare the following table with the reactants of the reaction:
Buffer _______ul
DNA ______ug _______ul
Enzyme ______U _______ul
Water _______ul
Given,
- Alu 1 enzyme concentration: 5,000 U/ml
- Alu 1 enzyme buffer concentration: 10X
- DNA concentration: 4 ug/ul
- Desired DNA amount: 12 ug
- Desired total volume: 25 ul
- Enzyme to DNA ratio: 1 U/ug
Question A
1. Enzyme Volume (ul):
- Enzyme needed (U) = DNA amount (ug) × enzyme to DNA ratio (U/ug) = 12 ug × 1 U/ug = 12 U
- Volume of enzyme (ul) = Enzyme needed (U) / Enzyme concentration (U/ml) = 12 U / 5,000 U/ml ≈ 0.0024 ml ≈ 2.4 ul
2. Buffer Volume (ul):
- Since the buffer is 10X concentrated, we'll need to dilute it with water to make it 1X. So, buffer needed (ul) = Total volume (ul) / Buffer dilution factor = 25 ul / 10 = 2.5 ul
3. DNA Volume (ul):
- Volume of DNA needed (ul) = Desired DNA amount (ug) / DNA concentration (ug/ul) = 12 ug / 4 ug/ul = 3 ul
4. Water Volume (ul):
- Total volume - (Enzyme volume + Buffer volume + DNA volume) = 25 ul - (2.4 ul + 2.5 ul + 3 ul) ≈ 17.1 ul
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