BIO306 Lab

• what does the X-Amp do in our DNA extraction?

  X-Amp reagent helps denature the components of the fungal cell wall, cell membrane, nuclear membrane, and proteins. This allows the release of chromosomal DNA into the solution.
• why were our PCR primers tagged?

  to be able to distinguish which PCR product came from which table
• what is DNA barcoding?

  using short DNA fragments from a specific genetic locus and comparing that sequence to sequences in a database in order to identify an organism
• what region of DNA was amplified for barcoding? why was this region amplified?

  for our fungal identification, the ITS locus was amplified. this region was amplified because is was approved by the International Barcode of Life Consortium. The standardization of loci used allows anyone to obtain genetically comparable data by using the same PCR primers.
• why did we use magnetic beads to purify our PCR products?

  this protocol uses SPRI (solid phase reversible immobilization) beads, which are like magnetic particles with carboxyl groups that will reversibly bind DNA based on its surroundings. The beads essentially promote DNA binding, so the DNA will bind to the beads, leaving the impurities in the solution.
• what is a good 260/280 absorbance ratio for a DNA sample?

  1.8
• what is a good 260/230 absorbance ratio for a DNA sample?

  2.0
• how does nanopore sequencing distinguish between the 4 DNA bases?

  the nanopore sequencing machine records the electric current when the DNA is pulled through the nanopores. Certain electrical currents correspond to a specific base in the DNA
• what does the E value from a BLAST search tell us?

  the likelihood that your sequence and the sequence in the table aligned by random chance
• is a low or high E value more precise?

  low
• what does % query cover tell us?

  the length of our sequence compared to the reference sequences provided by the BLAST search (100% coverage would indicate that out sequence spans the entire length of the sequence provided by BLAST)
• what does % identity tell us?

  the percent of nucleotides or amino acids that are identical between our sequence and the reference sequence provided by BLAST