Bio Lab Final

• ELISA process

  Antigen → primary antibody → secondary antibody → conjugated enzyme --> substrate
• Positive ELISA result

  Positive color shows to be blue (substrate binds to conjugated enzyme of secondary antibody showing the existence of antigen in the well)
• Negative ELISA result

  Negative control shows that nothing is bound in the reaction, and shows a clear color indicating no antigen there
• Negative and positive controls

  Positive control shows the existence of antigen, negative control shows no antigen
• Functions of negative and positive controls

  To show whether a substrate has binded to a secondary antibody, which would mean that there was an antigen in the well
• Interpreting ELISA results

  1. Blue will be positive - meaning the antigen binding has been detected

  2. Clear will be negative - meaning that there is no antigen
• Positive ELISA test


  Presence of serum antibodies against a specific pathogen indicates that the patient has encountered a disease + Immune system produces antibodies that go against antigen (foreign HIV). If antibodies from patient blood binds to antigen meaning patient has disease (Positive test would show color change and antigen existed)
• False negative ELISA result

  No binding interactions which could be because there is no antigen present. Due to no antigen present, there is no binding and the antibody is then washed out afterwards
• False positive ELISA result

  Mistakenly adding antigen, not exchanging pipettes which caused contamination.
• Purpose of PCR reaction

  To amplify small amounts of a DNA sequence of interest so it can be analyzed separately
• Essential components of a PCR reaction

  • Target DNA
  • A pair of primers specific for the target DNA
  • Free Deoxynucleotide
  • Heat stable DNA polymerase (Taq)
  • Buffer (containing cofactor Mg+)
• DNA template for PCR

  DNA that is specific to the primers that are being used can be used as a template. Both crude and pure DNA can be used because primers are specific and will attach to its designated DNA
• PCR primer properties

  Usually consist of a pair of primers (2 primers) in which both are roughly about 20 nucleotides in length and must be complementary to the 3' end of the gene to be amplified specifically for the gene. They are designed to flank the region to be amplified. Quantity: In large excess
• Purpose of primers in PCR

  In the absence of primers, DNA synthesis cannot occur. Once a primer is present, DNA polymerase can start at the desired location of the DNA strand and take over in synthesizing the new chain
• Polymerase enzyme in PCR

  Taq polymerase can survive at high temperatures (ideal at 75℃) therefore it can sustain undergoing multiple rounds of PCR which require high temperatures without being permanently denatured
• Basic steps in each PCR cycle

  1. Denaturation at 93℃ for 30 seconds which separates strands

  2. Annealing the primer at 58℃ for 30 seconds which is the rejoining of the 2 strands via Hydrogen bonding

  3. Primer extension at 72℃ for 30 seconds which helps the synthesis of new strands
• PCR amplification

  Exponential; can calculate from 2^n where n= # of rounds and will double at the end of each cycle
• Determining PCR product size

  The placement of the left and right primer. For example in a genome map, if the left primer is at 15,971 and the right primer is at 16,411, we would find the difference to find the PCR product
• Functions of dNTPs in PCR

  • Provides building blocks for new DNA strands to be copies during PCR
  • Function as an energy source for reaction
• Purpose of MgCl2 in PCR

  MgCl2 is included in the reaction because it is a cofactor for Taq polymerase and helps speed up overall reaction
• Setting up PCR reaction in lab

  Obtain PCR reaction tube (containing a small Ready-to-Go PCR bead)

  1. Use a micropipette (w/ fresh tip) to add 20 μl of the appropriate primer/loading buffer mix to the PCR reaction tube. Tap tube to dissolve bead (bead contains Taq polymerase)
  2. Use red fixed-volume micropipette and fresh tip to add 5.0 μl of human DNA to PCR reaction tube and tap to mix
  3. Label cap of tube with sign-in # and section #
  4. Place PCR reaction tube in ice bucket next to the PCR machine
• Analyzing PCR product

  The student needs to analyze their PCR product through the gel electrophoresis (the DNA ladder is also used in this process). Gel electrophoresis will show the student roughly how many base pairs are in his product when he compares it to the DNA ladder on the gel
• Basis of DNA separation in agarose gel


  Agarose gel separation in this lab relies on the size of the DNA fragment; smaller fragments travel further
• Negative charge of DNA


  Phosphate groups are negatively charged thus making DNA negatively charged (migrate to anode)
• Factors affecting DNA migration rate in agarose gel

  Molecular size of the DNA

  Conformation of DNA (circular vs linear)

  Agarose concentration

  Buffer

  Applied voltage
• Staining DNA in gel

  Ethidium bromide intercalates (binds and causes structural changes when binding) into DNA molecules and causes molecules to be fluorescent under UV lamp
• Purpose of marker lane in gel

  Marker lane allows for a general map for predicting and comparing where DNA molecules will migrate to by using a ladder with overall bp numbers labeled
• Relationship between DNA size and migration rate

  Bigger molecules have slower migration rates while smaller molecules have faster migration rates. Mobility of linear DNA fragments is inversely proportional to log10 of molecular weight
• Dyes in marker lane

  Bromophenol blue ("faster" dye) and Xylene cyanol ("slower" dye) both allow for visual monitoring under a UV lamp of how far the DNA fragments have traveled in electrophoresis
• Genetic transformation


  A process that introduces a specific gene into an organism and has that genetic information expressed by the organism
• Constitutive vs inducible gene expression


  Constitutive gene expression lacks regulation and are always on whereas inducible gene expression, the system is always off until the presence of a molecule
• Characteristics of E.coli strain and pGLO plasmid

  E.Coli bacteria and pGLO plasmid that codes for GFP and bla, which provides antibiotic resistance. pGLO can be used to control expression of fluorescent protein in transformed cells. E.Coli was used because it is a single celled organism that produces quickly and will not infect plants or animals and it cannot grow on plates with antibiotic (ampicillin)
• Purpose of LB/amp media

  LB/amp media is used for transformants because the LB becomes resistant to ampicillin when +pGLO is present
• Purpose of arabinose

  Arabinose helps activate Green Fluorescent Protein
• Plates that produced glowing colonies

  LB/amp/arabinose produced glowing colonies because the presence of arabinose activates GFP (green fluorescent protein)
• Control plates and their purposes

  LB plate w/o pGLO is a Positive Control whereas LB/amp plate w/o pGLO is a negative Control
• Calculating transformation efficiency

  Transformation efficiency = transformants/μg of plasmid DNA
• Transformation efficiency

  Transformants/μg of plasmid DNA
• Dilution


  Calculated by the formula C1V1=C2V2 where C1 represents the concentration and V1 represents the volume of stock solutions whereas C2 and V2 represents the concentration and volume of the dilute solution respectively

  
• Absorption spectrum


  Used to discover lambda max where x axis is wavelength (in nm) and y axis is absorbance (no units)