lab 5 Flashcards
what is the mini prep procedure?
The miniprep procedure separates plasmid DNA and some bacterial RNA from the bacterial chromosomal DNA, bacterial proteins and membranes.
what do we do after the mini prep procedure to further purify plasmid DNA?
Because the miniprep still contains bacterial RNA, it can be further treated with RNAse which degrades the remaining RNA without affecting the plasmid DNA. However, when large amounts of purified plasmid DNA are required, it can be separated from bacterial RNA by GEL FILTRATION CHROMATOGRAPHY
what are the 2 techniques used in this lab so separate plasmid DNA and bacteria RNA
In this lab you will be given a mixture of plasmid DNA and RNA which will first be separated into fractions using GEL FILTRATION CHROMATOGRAPHY. The fractions you collect from this procedure will then be further separated using AGAROSE GEL ELECTROPHORESIS. The combined procedures will result in a clearer separation of the nucleic acids
by what principle does gel filtration chromatography separate molecules?
size and shape
by what process does a bacteria acquire a foreign plasmid?
transformation
what do we need in order to introduce a foreign gene into a cell? what is it in this case?
a vector. the vector in this case is the plasmid
what are the 2 types of plasmid replication?
-stringent: occurs with the bacterial chromosome (one copy per cell)
-relaxed: occurs autonomosly (multiple copies per cell (10-500))
why is inserting genes into cells a useful practice?
-clone a gene to use in gene therapy
-encourage the cell to translate it into the gene product
how does the bacteria replicate the human gene once its in the bacterial cell?
because the gene code is univresal
what are the components of the gel filtration chromatography experiment?
matrix, chromatography column and the elution buffer
what part of the chromatography matrix actually performs the separation?
the matrix (the beads)
what is the stationary phase of the chromatography?
the matrix (beads)
what is the mobile phase of the chromatography?
elution buffer
what does the column of the chromatography matrix consist of
consists of a tube with a frit and elution spout
what’s the frit?
a membrane or porous disk that supports and retains the matrix in the column but allows water and dissolved solutes to pass
what does the elution buffer do?
The column, with the matrix and applied sample, is “developed” by the elution buffer. This means that the molecules in the sample are carried by the flow of buffer into the matrix where they are gradually separated.
what is recombinant DNA?
two sources of DNA together
what is bigger RNA or DNA?
DNA
what does the matrix consist of?
consists of microscopic beads that contain pores and internal channels. The larger the molecule, the more difficult it is for it to pass through the pores and penetrate the beads.
how does gel filtration chromatography separate molecules?
the larger, higher molecular weight molecules are eluted from the column before the smaller ones. Larger molecules take the faster, more direct path that involves less time in the beads
when do we conduct agarose gel electrophoresis? what is the point of it?
after gel filtration chromatography. will be used to analyze and further separate the RNA and DNA after chromatography
how does agarose gel electrophoresis separate molecules?
on the basis of their size and shape
what weighs more, plasmid DNA or RNA?
plasmid DNA
how does the agarose gel separate molecules?
The agarose gel has microscopic pores that act as a molecular sieve.
