PLASMID ISOLATION AND ANALYSIS Part II Plasmid Purification and Isolation

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PLASMID ISOLATION AND ANALYSIS Part IIPlasmid
Purification and Isolation
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Plasmid Purification and IsolationStep 1
Pellet 2 ml of overnight cell suspension by
adding 1 ml (20 drops) of overnight culture
suspension into a 1.5 ml reaction tube. Spin
for 1 minute at 10,000 rpm in a microcentrifuge
(MicroV).
PLASMID ISOLATION AND ANALYSIS Part II
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Plasmid Purification and IsolationStep 1 (cont.)
A pellet should form in the bottom of the tube.
Drain off the liquid into a designated container
and gently tap the tube on a paper towel to
remove the excess broth. Repeat the procedure
with a second 1 ml volume of overnight culture
suspension.
PLASMID ISOLATION AND ANALYSIS Part II
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Plasmid Purification and IsolationStep 2
Using a small needle pipette, completely
resuspend the cell pellet by adding the entire
contents of the tube marked Cell Resuspension
Solution(P1). Mix the contents by finger
flicking the tube or pipetting several times. No
visible clumps of bacteria should remain.
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Plasmid Purification and IsolationStep 3
Use a small needle pipette to add the contents of
the tube marked Cell Lysis Solution(P2) to the
Resuspension tube and mix by inverting the tube
4 times. The cell suspension should appear clear.
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Plasmid Purification and IsolationStep 4
Use a small needle pipette to add the contents of
the tube marked Neutralization Solution (N3)
to the Resuspension tube and mix by inverting
the tube 4 times.
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Plasmid Purification and IsolationStep 5
Centrifuge the tube containing the lysate at
10,000 rpm for 10 minutes.
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Plasmid Purification and IsolationStep 6
While the lysate is in the centrifuge, label two
1.5 ml tubes. Label the lid and side of one
tube L.
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Plasmid Purification and IsolationStep 7
Label the lid and side of the second tube M.
Remove the lid from the tube with a scissors.
Place the lid back on the tube and hold until
Part III, step 7.
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Plasmid Purification and IsolationStep 8
Use the small needle pipette to carefully remove
all of the clear lysate/supernate from the
miniprep in step 5. Do not remove any of the
precipitate. Wipe the end of the pipette with a
chem wipe or kleenex to remove any
protein/chromosomal DNA precipitate.
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Plasmid Purification and IsolationStep 9
Pipette the clear lysate/supernate into the tube
marked L. At this point you can choose to
store the tube in the refrigerator until the next
day or, if time permits, continue to Part III.
PLASMID ISOLATION AND ANALYSIS Part II