PCR + Gel electrophoresis Flashcards
what is the purpose of PCR
pcr is used to look for a particular gene/piece of dna that is present in the genes of many but not all
what is the objective the pcr lab
the objective is to produce large amounts of dna in a test tube starting from a trace amount
explain the basic procedures during the preparation period
once DNA is extracted, transfer a microtest tube containing the instagene matrix and proteases, then lyse the cells by heating it up to release all of the cellular constituents. Once the cells are ruptured, the DNases can can digest the released dna however when the cells are in the presence of the instagene micture the cofactor is absorbed and not available to the to the enzyme
explain the purpose of each the instagene mix
the instagene mix traps metal ions such as Mg2+ which are required as catalysts/cofactors in enzymatic activity
purpose of proteases
digests the connections between cells and allows for better lysis
define the instagene mix and proteases
they are made of neg charged microscopic beads that chelate (grab) metal ions out of a solution
what happens when we heat up to release cellular constituents
when this happens, not only is our dna released but also enzymes that were once contained in the cell lysosome such as dnases which is used by cells to digest the dna of invading viruses is extracted and released
what is possible after release of the DNases
the dnases can digest the released dna
what prevents the possibility of the DNases to release digest the dna
the cells are in the presence of the instagene beands in which the cofactor Mg2+, which speeds up Dnases ability to digest released dna, is absorbed and therefore not available to the enzyme; therefore virtually blocking enzymatic activity and degredation of the extracted dna so you can use it as a templet in the PCR
explain the heating procedures in the preparation period of PV92 s
1) suspend the dna molecules in the instagene mix with proteases and incubate at 56 C for ten minutes
2) place the cells in 100 C boiling water bath for five minutes
what does 56 C do to the dna
it softens the plasma membranes and releases clumps of cells from each other
what does 100 C do to the dna
this is where the cells ruptures and releases DNA from the cell nuclei
what is the purpose of PCR amplification
since PCR has the ability to make millions of copies of a
what is found in the master mix?
dna templet, individual deoxynucleotides, dna/taq polymerase, magnesium ions, olignucleotide primers, and a salt buffer
what is the purpose of each of the ingredients in the master mix
dna/taq polymerase is needed for assembly of the new dna, the mg ions are needed as a cofactor (catalyst) required by dna/taq polymerase to create the dna
