Lab aims

• SNPs (Single Nucleotide Polymorphisms) are variations in a single nucleotide (A, T, C, or G) at a specific position in the genome.

  Importance:

  • Act as genetic markers for locating genes linked to diseases.
  • Influence individual traits like disease susceptibility and drug response.
  • Help in studying evolutionary biology and genetic diversity.
• How do you prepare a buffer solution?

  1. Select a buffer system: Use an acid-base pair with a pKa near your target pH.
  2. Calculate proportions: Use the Henderson-Hasselbalch equation
  3. Mix components: Combine the weak acid and conjugate base in calculated amounts.
  4. Adjust pH (if needed): Add small amounts of acid or base.
  5. Dilute to desired volume.
• How does PCR work and what is it used for in molecular biology labs?

  1. Denaturation: Heat to separate DNA strands (~94°C).
  2. Annealing: Cool to allow primers to bind to target sequences (~50-65°C).
  3. Extension: DNA polymerase synthesizes new DNA strands (~72°C).
  4. Repeat for ~30 cycles to amplify the target DNA.

  • Amplify DNA for cloning, sequencing, or analysis.
  • Detect genetic mutations or pathogens.
  • Perform forensic DNA profiling.
  • Quantify gene expression (via qPCR).
• How is gel electrophoresis used to examine DNA?How it works:

  • DNA samples are loaded into a gel matrix (e.g., agarose).
  • An electric current pulls negatively charged DNA toward the positive electrode.
  • Smaller DNA fragments move faster and travel further, separating by size.

  Uses:

  • Verify DNA size and purity.
  • Analyze PCR products.
  • Identify genetic variations (e.g., RFLP).
  • Confirm successful cloning or sequencing.
• How and why are restriction enzymes used in labs?How they work:

  • Restriction enzymes cut DNA at specific sequences called recognition sites.
  • Cuts can produce "blunt" or "sticky" ends, enabling precise DNA manipulation.

  Why they're used:

  • DNA cloning: Insert DNA fragments into vectors.
  • Genotyping: Identify genetic variations.
  • Mapping DNA: Analyze genome organization.
  • Gene editing: Prepare DNA for further modification.
• Role of detergent = breaks the cell membrane
• Role of EDTA = prevents DNAses from breaking down the DNA
• Role of silica = Traps DNA into a column
• PCR mastermix

  • dNTPs = provide procursors for DNA synthesis
  • Taq DNA polymerase = amplifies specific sequences
• Nucleotide triphosphates are added to growing DNA strands during which stage = extension
• • Blastx = translates a user entered nucleotide query sequence to search for matches in the protein sequence
  • protein blast = uses a protein query sequence to search for matches in the protein database
• SNPs

  • synonymous = change in DNA sequence which doesnt lead to a change in amino acid coded for. no effect on protein
  • non-synonymous = leads to a different amino acid being coded for and may change protein structure and function
  • stop gain = non-sense, when a change in DNA sequence produces a STOP codon, early termination , non functional protein