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nick and hybridisation - Nick Translation Nick Translation...

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Unformatted text preview: Nick Translation Nick Translation: Involves the synthesis and labeling of a Probe A piece of Nucleic Acid labeled (incorporated) with a Tracer that allows one to "track" the hybridization of the Probe to an unknown sequence of DNA A Tracer could be: 1.) Radioactive element or isotope of an element (Ex: 32P) 2.) Macromolecule Ex: Digoxigenin (Dig) a large steroid molecule recognized by a specific Antibody (AntiDig) our chosen tracer To synthesize and label a Probe, We need the following components in a reaction mixture: 1.) dsDNA (double stranded DNA) dsDNA sequence must be known 2.) DigLabeling Mixture (Nick Translation Mixture) Composed of the following: a.) dNTP's (Deoxyribonucleotide triphosphates) P P P S NA dATP dGTP dCTP dTTP This has been replaced by: dUTP Digoxigenin b.) 10X Reaction Buffer c.) DNase I maintains the pH at neutral (pH = 7) an endonuclease that nicks (cuts) DNA randomly on one strand an enzyme that has 2 primary functions: 1. 5' 3' exonuclease activity* 2. 5' 3' polymerization (elongation) activity d.) DNA Polymerase I *utilizes this activity as part of its proofreading (errorchecking) activity dsDNA of known sequence 3' 5' 5' 3' DNase I randomly nicks this strand, as well as the opposite strand 3' 5' 3' polymerase activity 5' 5' 3' 5' 3' exonuclease activity dATP dCTP dGTP dUTP Dig DNA Polymerase I "sees" the nick and quickly utilizes it's 2 primary functions. It first removes between 35 nucleotides ahead of the cut, then fills in the gaps with the appropriate dNTP's Dig 3' 5' 5' 3' Labeled Probe Dig Dig Boiling Water Bath: dsDNA ssDNA (single stranded DNA) Hybridization of the Blot Key Points in the Hybridization of the Blot: Prehybridization Solution: blocks nonspecific DNA binding sites cuts down on "background" hybridization and enables one to see the bands (fragments) more clearly Washings (2): Low Stringency (2X Saline Sodium Citrate) removes nonspecific High Salt Concentration & Low Temperature (RT) binding (hybridization) High Stringency (0.2X Saline Sodium Citrate) Low Salt Concentration & High Temperature (65OC) Heating the Probe: denatures (melts) the doublestranded (ds) DNA into singlestranded (ss) DNA, allowing the annealment (base pairing) with the ssDNA on the nylon membrane (blot) Hybridization: annealing the probe to the target DNA on the nylon membrane HYBRIDIZATION OF THE BLOT: 1.) Get a plastic container from the front and label your container. 2.)Place your nylon membrane into your container, DNA SIDE UP! 3.) Pour 5mL of preheated prehybridization tube onto your membrane . 4.) Incubate for 15 minutes in 65 degree incubator. 5.) Boil your probe test tube DNA (get the test tube from me) in the 100 degree water block for 2 minutes. 6.) Once done boiling add 480 microLitre of room temperature pre hybridizing solution . 7.) Take nylon membrane out of incubator and drain out liquid. 8.) Add your 100 microlitre of the boiled probe DNA hybridization mixture to your nylon membrane (DNA side up still). 9.) Incubate for an hour in the 65 degree incubator! After 1 Hour Incubation: Pour liquid into a beaker. Add 10ml of low stringency buffer (front of room) and shake for 5 minutes. Pour off solution and add another 10ml low stringency buffer and shake for 5 minutes. Pour off solution. This time add 15ml of HIGH STRINGENCY BUFFER and place is it still for 15 mins. Pour off solution and store in your section drawer between labeled filters as before. ...
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