The DNA chain extends in a 5' to 3' direction and requires a pre-existing 3'-OH to continue the chain. ddNTP's terminate extension of the chain
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What needs to be added to the test tubes for dideoxy sequencing
DNA template, DNA primer, mixture of dNTPS, a small quantity of ONE ddNTP, and DNA polymerase
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Explain the difference in fluorescence automated sequencing
Each of the dideoxynucleotides are labelled with a different fluorescent dye, all four of the reactions take place in the same tube and can be loaded into the same lane of the gel. The colours are detected by a fluorimeter.
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Another sequencing method
Pyrosequencing, where the final phosphate is labelled and recorded as it released upon synthesis
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Describe the maxam and gilbert method
DNA fragments are subjected to random cleavage at ACGT positions using specific chemical agents. The DNA strand breaks after the base is modified and removed
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What is SDS page used for?
Separating a mixture of proteins and comparing expression between samples, both quantitavely and qualitatively
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What is the detergent SDS used for?
it binds to the backbone of proteins in an amount proportional to the size of the protein. This masks the charge and prevents protein aggregation.
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Why are the protein samples boiled in SDS page
to disrupt secondary structure. Hyrogen bonding and electrostatic interactions
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How do we remove Disulphryl bridges between protien subunits?
use B-mercaptoethanol in the sample buffer which reduces the S-S bridges
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The type of gel used - and the two phases
polyacrylamide gel, 2mm gel poured between two plates and run vertically. 4% stacking gel, gets all the protiens in a single band. 12%15% resolving gel, where the protiens are separated according to size
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Three dyes that can be used to visualise the protiens after separation
coomassie blue, silver, SPYRO orange
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Western blotting - what is it for?
to detect proteins in a sample of tissue homogenate by specific antibody interactions
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Explain the proceedure for western blot
an electric current transfers the protiens, that have been separated by SDS Page, to a porous membrane sheet. This is incubated with an antibody which sticks to the desired protien if it is present
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How can you identify which protein has been bound by the antibody?
Do an ELIZA - to activate colour reaction
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What problems can dimensional SDS page help us overcome
2 different proteins of the same size 'clumping together' and appearing as one highly expressed protein.
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Explain how 2-Dimensional SDS page works.
the SDS page is ran as normal, and then the gel is turned 90 degrees and ran again, this time with a different buffer allowing a pH gradient to be established.
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How can we determine which protien is in each spot?
purify the protien, sequence it by edman degredation.
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Edman degredation?
yes
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PCR. What is it for?
Amplify small amounts of DNA
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Describe the process
Denaturation, DNA primer annealing, extension. Taq polymerise is used
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What needs to be included for PCR to work
DNA primers, doxynucleotides, Mg2+ co-factor for enzyme, buffer, DNA polymerase - taq.
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Other cards in this set
Card 2
Front
Most common type of DNA sequencing
Back
dideoxy sequencing
Card 3
Front
Draw/describe dideoxythreonine
Back
Card 4
Front
Describe how dideoxy sequencing works
Back
Card 5
Front
What needs to be added to the test tubes for dideoxy sequencing
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