The Lab Flashcards

Question

Hypo-osmotic swelling test

Answer 1

Differentiates dead sperm from living sperm with an intact membrane that are immotile If living sperm is placed in a hypo-osmotic liquid, the sperm will swell to maintain an osmotic gradient resulting in swelling of the membrane and curling of the tail Normal if > 60% sperm swell in the sample Also useful during TESE because sperm become motile after passing through the epididymis

Answer 2

Modified golgi apparatus covering upper 75% of the sperm head between the inner and outer acrosomal membranes Contains hydrolases (most common acrosin) At time of acrosome reaction the plasma membrane and outer acrosomal membrane fuse (exocytic process)

Answer 3

Genetic disease leading to absence of the acrosome

Answer 4

~130 live births Primordial follicles/primary oocytes in the ovarian cortical tissue are frozen Thin layer of cortex is frozen (1 mm)

Answer 5

Metaphase of meiosis II with chromosomes arranged on bipolar spindle

Answer 6

Denaturation -> Annealing -> Extension/Elongation Denaturation: heat DNA (20-30sec) yields 2 ss DNA molecules @ 92 degrees Annealing: lower temp to 50–65 °C for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. One for each of the two single-stranded complements containing the target region. Extension/elongation: Raise temp @ 72 degrees, DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free nucleotides (dNTP)

Answer 7

Advantages: Rapid, fast, the sequencing of unknown etiologies of many diseases are being figured out by the PCR. Allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail (e.g. Down syndrome for only a few fetal cells) Disadvantages: One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification.

Answer 8

faster than conventional PCR Uses fluorescent probes to measure the amount of DNA produced while PCR is occurring Provides a quantitative comparison between samples by limiting the analysis to the stage where the reaction shows linear amplification Used in the diagnosis of infectious disease or to measure gene expression (when combined with reverse transcription)

Answer 9

Excess labelled hormone + unknown amount in sample and limited antibody Competition between labeled hormone and that in sample. Less bound labeled hormone means more in the sample

Answer 10

extent to which a measurement agrees with the actual value QC: standard assay curve

Answer 11

variability encountered when measuring same sample multiple times Expressed as coefficient of variation = SD/mean of replicate determinations x100 (%)

Answer 12

homodimeric glycoprotein linked by disulfide bonds TGF-B superfamily Typically measured by ELISA

Answer 13

switch from sandwich (ELISA assay) to competitive RIA

Answer 14

consider using elevated E2/P4 to suppress

Answer 15

The physical separation of components in a mixture via two phases Mobile phase: mixture is dissolved in fluid Stationary phase: substance is fixed in place Works because constituents of a mixture travel at different speeds, causing separation. Differential retention occurs in the stationary phase due to differences in a compound’s partition coefficient. Can be used to purify components in a mixture as well as to analyze for the presence of (or measure proportions) of analytes in a mixture.

Answer 16

Measures the masses within a sample via substance ionization and subsequent sorting of ions into a spectrum based on their mass-to-charge ratio Plot of the relative abundance of detected ions as a function of the mass-to-charge ratio is generated so that atoms or molecules can be identified via: Correlating known masses to identified masses Detection of a characteristic fragmentation pattern Often used in combination with chromatography to detect components of the mixture being separated The physical separation of components in a mixture via two phases Mobile phase: mixture is dissolved in fluid Stationary phase: substance is fixed in place Works because constituents of a mixture travel at different speeds, causing separation. Differential retention occurs in the stationary phase due to differences in a compound’s partition coefficient. Can be used to purify components in a mixture as well as to analyze for the presence of (or measure proportions) of analytes in a mixture.

Answer 17

1) Free nucleotides 2) Primers 3) DNA polymerase Taq = Heat-stable 4) DNA template (target) 5) Thermal cycler

Answer 18

Amplifies a sequence of RNA after it is converted to its complementary DNA by reverse transcriptase (originally discovered in retroviruses) cDNA is then processed by standard PCR procedure to yield large quantities of DNA corresponding to the RNA of interest

Answer 19

Set of approaches that allow the nucleotides in a particular portion of the genome to be identified in order Most common approach for the past 40 years was Sanger sequencing (also known as chain-termination sequencing) An in vitro DNA replication is carried out using DNA polymerase at sites directed by DNA primers Nucleotides include a subset that terminated DNA strand elongation- called dideoxynucleotides or ddNTPs ddNTPs are typically fluorescently labeled to allow automated detection with the fluorescent wavelength matched to the length of the fragment

Answer 20

21 cycles and 35 ET

Answer 21

caffeine or pentoxifylline can also be used where sperm are alive but not swimming and it works on sperm that has been frozen/thawed