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Biol435Group#3
Fanaye WoldeamanuelCarina Alvarez
Mary Carter
Jonathon Quintero
Laboratory #5 DNA Miniprep
Required Reagents/Supplies
MicrofugeEpendorf tubes
MicropipettesMicropipette tips
Miniprep solutionsQiagen DNA purification columns
Lab Outline/Timeline
Lecture/40 minutesDNA Purification/65 minutes
DNA Manipulation/60 minutes
Once the bacteria have taken up (been transformed) by the
plasmid, you may wish to re-isolatethe plasmid from the bacteria.
This could be done for many reasons but the two most likely are
to amplify the plasmid (increase the amount of plasmid
available) or to isolate a new plasmid (aswill be done later in the
semester). While DNA purification can be done without a
pre-prepared
kit, these methods tend to be as expensive and produce more
chemical wastes than the current
kits.
Protocol
1. Gently swirl the contents of the culture tube to resuspend
the cells.2. Pipette 1-1.5ml of solution into a 1.5 mL tube.3. Cap,
and place the tube in a centrifuge at maximum speed for 5
minutes.
a. This part of the procedure collects the bacterial cells which
are suspended in theliquid medium into a cell pellet at the bottom
of the tube.
4. Withdraw the tube from the centrifuge and discard the
supernatant using a pipettor.Note: Be careful not to remove all of
the supernatant
5.
Add 250 uL of Buffer 1 to each tube and resuspend the cells by
vortexing. Make surethat the cell suspension is homogenous and no
clumps are visible.Buffer 1 includes: 50 mM Tris-HCl, 10 mM EDTA,
100 ug/mL RNase A, pH 8.0
b. The cells are now resuspended in a buffered solution with
RNase. When the cellsare lysed in the next step, the RNase will
catalyze hydrolysis of all RNA
molecules into nucleotides, but the DNA will not be affected.6.
Add 250 uL of Buffer 2 to each tube. Close the caps and mix the
solutions by gently
inverting the tubes 5-6 times.
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NOTE: DO NOT VORTEX since the chromosomal DNA released from the
broken cells
could be sheared into small fragments and contaminate your
plasmid prep.
Buffer 2 includes: 1% SDS, 0.2 M NaOH
c. SDS is an acronym for Sodium Dodecyl Sulfate. It is an ionic
detergent whichdisrupts cell membranes and destabilizes all
hydrophobic interactions holdingvarious macromolecules in their
native conformation. The high pH of the 0.2 M
NaOH also denatures macromolecules by changing the condition of
ionizablegroups (ionizing certain groups and deionizing others).
The clearing you see is
because the cells are lysing. The viscosity of the solution is
increased by theincrease in concentration of macromolecules in
solution (a result of the cell lysis).
7. Let tubes sit in ice for 5 minutes.8. Add 350 uL of ice-cold
Buffer 3 to each tube. Again, close the caps and mix the
solutions
by rapidly inverting them a few times. A white precipitate will
form. Buffer 3 includes: 3.0 M Potassium Acetate, pH 5.5
d.
This is really the key step in the alkaline lysis procedure. The
low pH of thePotassium acetate solution neutralizes the NaOH and
when the pH returns to near-
neutrality then the macromolules renature. The proteins and
large DNAmolecules do not renature correctly however. They form
hydrophobic, ionic and
hydrogen bonds with each other nonspecifically because the
correct conformationof the molecule was not maintained during
denaturation. The plasmid DNA
molecules, however, never really fully denatured because they
are small circularmolecules which are supercoiled. Even though the
hydrogen bonds between base
pairs were broken by the high pH, they reform correctly when the
pH islowered. The large DNA molecules (chromosomal DNA) and
proteins form
precipitates because they bind to each other in a large
aggregate but the plasmids
don't precipitate because they renature correctly and don't
become part of thelarge multi-molecule aggregates. Thus plasmid DNA
remains in solution whileproteins and other DNA molecules
precipitate.
9. Let tubes stand on ice for 5 minutes.10.Place the tubes in a
centrifuge and spin at maximum speed for 10 minutes. A
precipitate
will form along the side of the tube.11.Place QIAprep spin
column in a 2ml collection tubes.12.Apply the supernatants from
step 7 to the QIAprep column by pipetting13. Centrifuge 30-60 sec.
Discard the flow through14.Wash QIAprep spin column by adding
0.75ml of Buffer PE and centrifuging 30-60 sec.15. Discard the
flow-through, and centrifuge for an additional 1min to remove
residual wash
buffer16.Residual wash buffer will not be completely removed
unless the flow-through is
discarded before this additional centrifugation. Residual
ethanol from buffer PE mayinhibit subsequent enzymatic
reactions.
e. Place QIAprep column in a clean 1.5 ml microfuge tube. To
elute DNA, add 50ul of Buffer EB (10 mm Tris-Cl, pH 8.5) or H20 to
the center of each QIAprep
column, let stand for 1min, and centrifuge for 1 min. (TE buffer
is commonlyused to redissolve DNA because it contains EDTA. The
EDTA will chelate
