biotechniques and experiments needed Flashcards
Griffith experiment (1928)
AIM: observe and prove the transformation principle
2 strains of strep used:
1. R strain: non virulent
2. S strain: virulent due to polysaccharide capsule that protects them from immune system
EXPERIMENT: creation of 4 mice
1.R strain mice lived
2.S strain mice died
3. heat killed S strain mice lived
4. !!! mix of heat killed S strain and live R strain mice DIED
CONCLUSION: transformation principle - a molecule/some material was exchanged between killed S and live R cells that transformed R cells and made them virulent. ALSO TRANSFORMATION IS HEREDITARY
Avery experiment (1944)
AIM: identify whether RNA/DNA/proteins was the transforming factor
EXPERIMENT:
-removal of lipids and carbs from sample so only DNA/RNA/proteins were present
-preparation of 4 tubes using S strain cell extract:
1. control tube containing all 3
2. tube with protease (no proteins) –> only DNA/RNA
3. tube with DNAse –> only RNA/proteins
4. tube with RNAse –> only DNA/proteins
!!introduction of R strain bacteria in each of the tubes
CONCLUSION: DNA is the transformation principle and carrier of cell genetic material because transformation only failed to occur in the tube containing DNAses
Hershey and Chase experiment (1952)
AIM: use of bacteriophages to prove whether proteins/DNA are inserted into cells in transformation (further proof of what was shown by avery)
EXPERIMENT: two sets of bacteriophages cultivated:
1. in radioactive sulfur medium
2. in radioactive phosphorus medium
-infection with E coli
-separation by centrifugation
CONCLUSION:
-Ecoli injected with S35 phages were not radioactive meaning the proteins were not incorporated in bacterium
-Ecoli injected with P32 phased WER radioactive meaning DNA was incorporated into cell
HENCE: DNA is the transformation principle
CHARGAFF experiment and conclusion
-used TLC to separate the bases of DNA
CONCLUSIONS: (4)
1. 4 nucleotides are not present in equal amounts
2. different species have different nucleotide content
3. A=T and C=G
4. cells making up tissues within organism share the same nucleotide content
diameter of DNA
2nm
!!!! stays constant because a purine is always being paired with a pyrimidine and so their combined length doesn’t change
how many base pairs can fit in one period of DNA
10.5
(period = same point going down helix)
why is RNA a lot more unstable and reaction
OH on carbon 2 of the ribose sugar opens itself to attack
Fast vs slow renaturation of DNA
FAST: rapid cooling of denatured DNA –> lower probability of collision between complementary bases on strands –> increases hybrids that contain irregular base pairings –> LIMITED RENATURATION
SLOW: gradual temperature cooling of DNA –> higher probability of correct collisions so produces highly/fully renatured strands
Relationship between extent of DNA denaturation/renaturation and absorption using photospectrometry
Absorption increases as denaturation increases (due to destruction of nitrogenous rings present in double helix which exposes the bases more)
HENCE:
single stranded DNA has highest absorption, fast renatured DNA has mid absorption, slow renatured DNA has low absorption
spectrophotometry
used to determine concentration of DNA due to direct proportion
!!SOS: RNA has a higher absorbance than DNA because it is single stranded
Gel electrophoresis of DNA
-separation of DNA fragments based on their size/molecular weight
-uses agarose gel with electrolyte solution and an electrical supply
RNA electrophoresis
-requires denaturing gel (containing formaldehyde) to eliminate any secondary folding
CHARACTERISTICS OF A SAMPLE THAT IS INTACT:
1. 28S and 18S prominent bands of the rRNA (most abundant)
2. slight smear of mRNA between them
protein electrophoresis
-SDS PAGE: sodium dodecyl sulfate polyacrylamide gel
-proteins denatured and boiled in SDS, which breaks 3ary structure, prevents reformation of higher structure and coats with negative charges
! reducing agent added for breaking of covalent bonds like S-S
-molecular weight in daltons
restriction enzyme use
-hydrolysis of phosphodiester bonds in specific location without the use of ATP
-sticky ends created
