Experiments/Procedures!! 💀😭 Flashcards
1
Q
Autoradiography and Cairns technique (aim, procedure, results)
A
Aim: Measuring the length of DNA chromosomes
Procedure:
- grow cells in radioactive thymidine so it is incorporated into the DNA of the cell
- Gently lyse cells so the chromosomes can be fixed on radioactive film
- the film is submerged in radioactively-sensitive silver bromide solution
- the radiation from the thymidine turns AgBr into solid silver grains
- excess AgBr is washed away –> silver grains appear as small black dots on the film
- Once developed, chromosome shape can be visualised with an EM
Advantages/results:
- measuring of chromosomes when uncoiled –> more accurate length
- using uracil, regions of active transcription can be identified
- showed the formation of replication bubbles in prokaryotes during chromosome replication
- bi-directional replication in the bubble (prokary)
2
Q
Mendel’s experiments (inheritance and cross-breeding)
A
- He crossed 2 purebred varieties of peas (tall and short) –> resulting in progeny which all had the same 1 trait
- Then he crossed the resulting progeny (self replication) and got the traits in a 3:1 ratio
- he performed these experiments many times to establish this ratio
Conclusions:
- a single factor controlling the trait (now called a gene)
- 2 versions of that factor (now called alleles)
- both parents contribute equally to the expression of the factor
- one version is more dominant, and will be expressed if present
- each gamete ha sonly one version of the factor (hence gametes are haploid)
The 3 laws:
- Principle of dominance: dominant allel traits always expresssed over recessive oens
- Law of segregation: in sex cells, alleles are separated so that each cell gets only 1 allele of the gene
- Law of independent assortment: The segregation of alleles for one gene occurs independently to that of any other gene
3
Q
Thomas Hunt Morgan and Drosophilia (fruit flies)
A
- he crossed red-eyed fruit flies with white eyed mutants and realised that the ratios didn’t conform to Mendelian ratios
- Red eyed males always produced red eyed females (red was dominant), but white eyed females always produced white-eyed males (it was x linked, so no x from father to mask the recessive trait)
- so he concluded that it was sex-linked
- Mans performed more crosses and realised that some phenotypes occured at ratios way lesser than mendel’s
- so he concluded that gene linkage occurred and some phenotypes wouldn’t form as the genes couldn’t independently assort
- if crossing over occurred to allow some phenotypes to exist –> occurred less frequently, hence the instances of those phenotypes were fewer
- genes with greater distance between them are more likely to cross over than those with lesser distance (think of it at more potential points on the chromosome for a chisama to form)
4
Q
PCR Stages, times and temperatures
A
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
Once large quantities of DNA have been created, other laboratory techniques are used to isolate and manipulate the sequences
5
Q
Gel electrophoresis
A
- Lab technique used to separate/isolate proteins or DNA fragments based and mass/size
- an electric current is used to move fragments through a gell medium, where smaller ones travel farther as they movement isn’t impeded by gel matrix as much
DNA:
- the DNA is cut by restriction endonucleases at different points, generating fragments
- the phosphate group gives DNA a negative charge, and when current is passed through, the fragments separate
- the fragments are put on an agarose gel and the distance they move is matched to industry standards to identify the fragment size
Proteins:
- proteins are folded into various shapes and may not have an equal charge across the entire structure
- the protein is anionised –> this gives it a linear structure and a constant negative charge across the protein –> allows the proteins to separate
- proteins put on polyacrylamide gel and the sizes are compared to known industry standards
6
Q
Leptin experiments on mice
A
- Surgically fusing the blood circulation of healthy and obese mice (parabiosis)
- Obese: either lack of leptin production or lack of receptors
- Lack of leptin production w Normal: The obese lost weight (leptin [] increased)
- Lack of functional receptors w Normal: Obese mouse didn’t gain or lose, Normal mouse lost as obese mouse blood had higher leptin –> lower appetite
Leptin use in humans:
- Mostly obesity caused by lowered receptor sensitivity –> increased leptin content has no impact
- side effects make it not worth it
7
Q
William harvey and sexual reproduction
A
- Studied sexual reproduction in deer
- observed the sexual organs of deer at different days after mating
- he concluded that the embryo was not formed from sperm mixing with menstrual blood, bc he wasn’t able to detect an embryo for 6-7 weeks post mating
8
Q
Electron tomography
A
- Sample is repeatedly imaged using a TEM
- Imaged at varying angles relative to the e- beam
- images compiled to forma. 3d representative image
- Samples must be prepped by ‘fixing and dehydrating’ or ‘freeze-drying’ –> stabilises structure –> ensures it doesn’t explode bc of the aqueous contents expanding and exploding
Features of mitochondria identified via this technique:
- cristae are continuous with the inner membrane
- even IMS throughout mitochondrion
- the shape, position, volume of inner matrix can change
9
Q
Calvin figuring out his (life) cycle (Lollipop experiment)
A
- Grew algae in this lollipop apparatus thing in the presence of radioactive C-14
- shone light on it to the c-14 got incorporated into the different organic compounds synthesised via photosynthesis
- after various time periods, killed algae by running it through hot ethanol (to halt metabolism)
- used 2d chromatography to separate the compounds
- used autoradiography to figure out which samples were radioactive
- used diff time periods to figure out the order in which reactions take place.
