Preparation and characterization of Plasmid DNA
Their restriction maps, or patterns of restriction endonuclease cutting can
characterize DNA molecules. Restriction endonuclease is enzymes that make double
stand cuts in DNA at specific sequences. The recognition sequence range from four to
eight base pairs.
A restrictions map is a diagram depicting the positions of the restrictions endonuclease
recognition sites and the distance betweens the sites, for a particular DNA molecules or
fragments of DNA. Restrictions maps are constructed by digesting DNA molecule with
different combination of restrictions endonucleses, then analyzing the digestion products
by gel electrophoresis in order to measure the lengths of the fragment.
Recombinant DNARecombinant DNA is formed by the joining two or more pieces of DNA from
different sources. The DNA is generally cut into fragments b restriction endonuclease.
Restriction endonucleasee cleavage overhanging , or sticky ends, which allow two
fragments to associate by base pairing ; the fragments can then be joined by the DNA
ligase . For instance , cutting with Bam HI leaves fragments with the ends-
--------G
and G-A-T-C-C--------
-----------C-C-T-A-G
G-------
Genes can be cloned by digesting chromosomal DNA with restriction endonuclease , then
ligating the resulting DNA fragments with another DNA molecule , a cloning vector.
Cloning vectors are small DNA molecule , such as viruses or plasmids , which are
capable of replicating within cells.
The plasmid pUC18 is a common cloning vector that can replicate extra chromosomally
in the bacterium Escherichia Coli. It contains genes for ampicillin resistance and lactose
fermentation., which allow cells carrying it to be identified. It also has a short stretch of
DNA , called polylinker, which contains sites for several different restriction
endonucleae.
The plasmid DNA samples are plasmid pUC18-HIS3 and pUC18-URA. PUC18-HIS3
contains HIS3 gene, excised from yeast genomic DNA by digestion with BamHI and
ligated into the BamHI of pUC18. PUC18-URA3 contains the URA gene, cut out the site
of genomic DNA with the restriction endonuclease HIndIII and ligated into HindIII site
of pUC18.
Purify Plasmid from Bacteria1) Grow cells in the AMP medium
2) Pellet cells / Centrifuge
3) Break open cell membrane4) Ethanol precipitation
4) Wash
Preparation of Plasmid DNA by alkaline lysis1) I resuspended the bacterial pellet in 400 uL of ice cooled solution I ( 50 uM
glucose, 25mM Tris-HCL, 10mMEDTA, pH-8) by vortexing vigorously.
2) Then, I mixed 400 uL of solution ( .2 M NaOH , 1% sodium dodecyl sulfate).
3) After that 400 uL of ice cold solution ( potassium acetate, 3M in potassium, 5M in
acetate)
4) Then centrifuge 5 minutes. I transferred the supernatant by pipet and discard the
pellet.
5) Then I vortex the solution by an equal volume of phenol:chloroform:isoamyl
alcohol(25:24:1)
6) I again centrifuge .
7) I gave two volume of ethanol to the precipitate and centrifuge again. I decant the
supernatant.
8) Then I added 70% ethanol and centrifuge for 5 minutes.
9) I resuspend the pellet in 50 uL TE buffer ( 10mM Tris –HCl, 1mM EDTA, pH 8)
Restriction endonuclease digestionBamHI, HindHIII, and PstI digested each DNA preparation for a total six times.
Agarose Gel electrophoresisThe DNA fragments produced by restriction endonuclease were separated by according
to size and shape by agarose gel electrophoresis.
Standard CurveI used a ruler to measure the each fragments of DNA, which has migrated from the well.
Then, I constructed a plot with the migration distance, in mm , on the X-axis, and the
logarithm of the DNA fragments length in base pairs on the Y-axis.