BIO577 Plasmid Purification and Analysis Laboratory Exercise
Overview
In this stage, you will analyze plasmids that have been successfully propagated to verify
that they have the RT-PCR product inserted. Purifying the plasmid from a small culture
of transformed bacteria (a miniprep) produces a sufficient amount of plasmid DNA. Next,
the plasmid is analyzed by restriction enzyme digestion to assess the size of the fragment
inserted and to compare it to the size of the RT-PCR product inserted into the plasmid.
Introduction
The QIAprep Miniprep system provides a fast, simple, and cost-effective plasmid
miniprep method for routine molecular biology laboratory applications. QIAprep
Miniprep Kits use silica membrane technology to eliminate the cumbersome steps
associated with loose resins or slurries. Plasmid DNA purified with QIAprep Miniprep
Kits is immediately ready for use. Phenol extraction and ethanol precipitation are not
required, and high-quality plasmid DNA is eluted in a small volume of Tris buffer
(included in each kit) or water.
Protocol: Plasmid DNA Purification Using the QIAprep Spin Miniprep Kit
This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from
1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium.
1. Pellet bacterial cells by spinning 1.7 ml of overnight culture for 1 minute at high
speed in a microcentrifuge.
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1.
Ensure that RNase A has been added to Buffer P1. No cell clumps should be
visible after resuspension of the pellet. The bacteria should be resuspended
completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
Mix gently by inverting the tube. Do not vortex, as this will result in shearing of
genomic DNA. If necessary, continue inverting the tube until the solution
becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for
more than 5 min.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube
4–6 times.
To avoid localized precipitation, mix the solution thoroughly, immediately after
addition of Buffer N3. The solution should become cloudy.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
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A compact white pellet will form.
6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or
pipetting.
7. Centrifuge for 30–60 s. Discard the flow-through.
8. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for
30–60 s. Discard the flow-through.
This step is necessary to remove trace nuclease activity when using endA+ strains
such as the JM series, HB101 and its derivatives, or any wild-type strain, which
have high levels of nuclease activity or high carbohydrate content. Host strains
such as XL-1 Blue and DH5™ do not require this additional wash step.
9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
10. Discard the flow-through, and centrifuge for an additional 1 min to remove
residual wash buffer.
Important: Residual wash buffer will not be completely removed unless the
flow-through is discarded before this additional centrifugation. Residual ethanol
from Buffer PE may inhibit subsequent enzymatic reactions.
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA,
add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAprep
spin column, let stand for 1 min, and centrifuge for 1 min.
Experimental Procedure for Restriction Digestion Analysis
Our RT-PCR product is cloned between two restriction sites in the pENTR/D-TOPO
vector: NotI and AscI. Unfortunately, these two enzymes are not compatible in the same
buffer, therefore we need to perform sequential restriction digests (with a purification
step between the two digests).
12. Label one microcentrifuge tube with your initials and plasmid number.
13. Prepare a digest for each of the plasmid preps as follows:
20 l plasmid prep
6 l dH2O
3 l NEBuffer 3
1 l Not I
*Incubate at 37oC for 1 hour.
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14. While the plasmid preps are incubating, prepare a 1% agarose gel. Each group will
need 5 lanes, so you may want to team up so that there is 1 gel/2 groups.
15. After incubation, purify the plasmid digest using the PCR purification kit from
QIAgen (you should all ready have the protocol for this). For the final step of the
purification process, resuspend you purified prep in 20 l of buffer rather than 50 l.
16. Set up the subsequent digest as follows:
20 l plasmid prep
6 l dH2O
3 l NEBuffer 4
1 l Asc I
*Incubate at 37oC for 1 hour.
17. We will run the gel on this double restriction digest on Thursday May7th.
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