Download Laboratory 11 PIPE Mutagenesis: Mini

Laboratory 11
PIPE Mutagenesis: Mini-prep and Sequencing
Concepts:
 DNA plasmid purification by mini-prep
 DNA sequencing
Goals: The goal of the lab this week is to finish the PIPE mutagenesis process. This involves
purifying the plasmid DNA from a few clones (colonies) and sending that DNA for sequencing.
You will mini-prep the plasmid, quantify the DNA, and send an appropriate sample to GeneWiz
for sequencing.
I. Introduction
The method used to purify nucleic acids is dependent on the type of DNA that you wish to
purify. There are unique methods to purify chromosomal DNA, plasmid DNA, mitochondrial
DNA, viral DNA, and RNA. Purification of plasmid DNA is via an alkaline lysis mini-prep
procedure.
The mini-prep procedure is used to extract plasmid DNA from bacterial cell suspensions and is
based on the alkaline lysis procedure developed by Birnboim and Doly (Nucleic Acids Research
7:1513, 1979). The procedure takes advantage of the fact that plasmids are relatively small
supercoiled DNA molecules. In contrast, bacterial chromosomal DNA is much larger and less
supercoiled. This difference in topology allows for selective precipitation of the chromosomal
DNA and cellular proteins from plasmids and RNA molecules.
In the procedure, the cells are lysed under alkaline conditions, denaturing both nucleic acids and
proteins. The solution is then neutralized by the addition of potassium acetate at a lower pH.
Because they are very large, the chromosomal DNA and proteins are unable to renature and
precipitate. Plasmids are small and are able to renature correctly and stay in solution, effectively
separating them from chromosomal DNA and proteins.
Like many things in molecular biology, companies have created kits to make routine lab
techniques easier, faster, more reliable, and (sometimes) cheaper. Today you will be using the
Qiagen Qiaprep Spin Miniprep kit. When performing a protocol from a kit, often times it is
unclear what each step of the protocol does. Below is a copy of a “traditional” alkaline lysis
protocol with descriptions of what each step accomplishes. The major difference between the kit
and the traditional method is that the kit uses a solid phase extraction to trap the DNA while the
traditional protocol uses an ethanol precipitation step to pull the DNA from solution.
Alkaline lysis mini-prep
1. Grow a 2.5 mL overnight culture
2. Label two 1.5 mL tubes and pipet 1250 µL of the cell suspension into each tube.
3. Close the caps and place the tubes in a microcentrifuge and spin at maximum speed for
20 s.
This part of the procedure collects the bacterial cells which are suspended in the liquid
medium into a cell pellet at the bottom of the tube.
4. Pour off the cleared supernatant.
5. Add 100 µL of Buffer 1 (50 mM Tris-HCl, 10 mM EDTA, 100 µg/mL RNase A, pH 8.0 )
to each tube and resuspend the cells by vortexing. It's very important that the cell
suspension is homogenous and no clumps are visible.
The cells are now resuspended in a buffered solution with RNase. When the cells are
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 200 µL of Buffer 2 (1% SDS, 0.2 M NaOH ) to each tube. Close the caps and mix
the solutions by rapidly inverting them a few times. DO NOT VORTEX. Vortexing
would break up the chromosomal DNA and contaminate your plasmid prep.
SDS is an ionic detergent which disrupts cell membranes and destabilizes all
hydrophobic interactions holding various macromolecules in their native conformation.
The high pH of the 0.2 M NaOH also denatures macromolecules by changing the
condition of ionizable groups (ionizing certain groups and deionizing others). The
clearing you see is because the cells are lysing. The viscosity of the solution is increased
by the increase in concentration of macromolecules in solution (e.g. DNA), a result of the
cell lysis.
7. Incubate on ice for 5 minutes.
8. Add 150 µL of ice-cold Buffer 3 (3.0 M potassium acetate, pH 5.5 ) to each tube. Close
the caps and mix the solutions by rapidly inverting them a few times. A white precipitate
will form.
This is really the key step in the alkaline lysis procedure. The low pH of the potassium
acetate solution neutralizes the NaOH, returning the pH to near-neutrality, allowing the
macromolecules to renature. The proteins and large DNA molecules do not renature
correctly, however. They form hydrophobic, ionic and hydrogen bonds with each other
nonspecifically because the correct conformation of the molecule was not maintained
during denaturation. The plasmid DNA molecules, however, never really fully denatured
because they are small circular molecules which are supercoiled. Even though the
hydrogen bonds between base pairs were broken by the high pH, they reform correctly
when the pH is lowered. 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 the large
multi-molecule aggregates. Thus plasmid DNA remains in solution while proteins and
other DNA molecules precipitate.
9. Incubate on ice for 5 minutes.
10. Place the tubes in a centrifuge (balanced) and spin at maximum speed for 5 minutes. The
flocculent precipitate will pellet along the side of the tube.
11. Transfer the supernatants into clean 1.5 mL tubes, being careful not to pick up any of the
precipitate. Discard the tubes with the precipitate and KEEP the tubes with the
supernatant (plasmid DNA is in solution).
12. To each tube of supernatant add an equal volume (about 400 µL) of ice cold isopropanol
to precipitate the nucleic acids. Close the caps and mix vigorously. Incubate for 2 minutes
then place them, with their hinges pointing outward from the center, in a centrifuge and
spin at maximum speed for 5 minutes. This step pellets the nucleic acids.
This concentration of isopropanol will cause the DNA to become insoluble. Since DNA
molecules are ionic (uniform negative charge due to the phosphates) they are highly
soluble in water but not soluble in organic solvents. Isopropanol and ethanol are
commonly used to precipitate DNA from aqueous solution.
13. The reason you oriented the tubes with the hinges outward is because the plasmid DNA
pellet may be difficult to see. It will be at the bottom and along the hinge side of the tube.
Carefully remove and discard the supernatant. The pellet is usually visible at this point. If
not, do not despair. It may be too small to see but there is probably enough DNA there.
Remember that we are dealing with small molecules that we can't see unless there is a
whole lot of them together!
14. Add 200 µL of absolute ethanol to each tube and mix by inversion several times.
15. Spin the tubes at maximum speed in a centrifuge for 2-3 minutes (hinges out).
This "ethanol wash" will speed up the process because the next step is to evaporate the
alcohol used in the precipitation step. Absolute ethanol evaporates much faster than a
solution of 50% isopropanol.
16. Carefully remove and discard the supernatant. Try to get as much out as possible without
dislodging the pellet of plasmid DNA.
17. Place the tubes in the fume hood with the caps open for 15-20 minutes to dry off the last
traces of ethanol.
18. When the ethanol is gone add 20 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH
8.0) to dissolve the pellet. Pipet the 20 µL in and out, up the side of the tube to ensure
that all of the plasmid DNA comes into contact with the TE buffer.
TE buffer is commonly used to redissolve DNA because it contains EDTA. The EDTA will
chelate magnesium ions which are a cofactor for most nucleases (enzymes which degrade
nucleic acids). If your DNA prep becomes contaminated with a nuclease (like the ones
produced by the cells in your skin) then the nuclease will be inactivated by the fact that
the magnesium cofactor is unavailable in the solution (because it is chelated by the
EDTA).
19. Store the pure DNA in the freezer.
Both protocols, the one you will follow for the Qiagen kit and the one above, start with an
overnight cell culture. This overnight culture will need to be prepared the evening before your
lab.
The Qiagen system of plasmid DNA purification employs a column based nucleic acid
purification system. The column contains a solid phase (silica) which the DNA binds to under
appropriate pH and salt conditions. The column is then washed to remove contaminants. Finally,
the pure DNA is eluted in a simple buffer or ddH2O. This type of DNA purification is often
called solid phase extraction. Solid phases can be anion exchange resin or silica. The choice of
the solid phase is dependent on the size of the DNA being purified and the downstream
requirements for the sample (e.g. avoiding reagents that interfere with subsequent steps).
DNA sequencing
DNA sequencing methods are used to determine the precise sequence of nucleotides in a stretch
of DNA. In this way, we can sequence the mutants obtained after PIPE mutagenesis to confirm
the mutation you sought to create. While DNA sequencing was once performed by individual
researchers using radioactive dNTPs, advances in the technique have led to core facilities and
companies that perform DNA sequencing quickly and inexpensively.
The classic method of sequencing, used to sequence the human genome in 2001, is the Sanger
method (Sanger F, Nicklen S, Coulson AR; Nicklen; Coulson. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1977. 74 (12):5463–7). This method takes
advantage of early termination of in vitro DNA replication. A set of four reactions are prepared
containing DNA template, a DNA primer, DNA
polymerase, a cocktail of four dNTPs, and a single
radioactively labeled modified di-deoxynucleotide
triphosphate (ddNTP). This ddNTP lacks the 3’ OH
group required for the phosphodiester bond between two
nucleotides. Once incorporated, the ddNTP prevents
further elongation of the DNA. The concentration of the
labeled ddNTP is set so that terminating nucleotides are
incorporated at a low enough rate to get a percentage of
each reaction terminated at each base. By running the
resulting DNA fragments from all four radioactive
reactions on a gel, the sequence can be determined
(Figure 11.1).
More recently, ddNTP have been fluorescently labeled
(Smith LM, Sanders JZ, Kaiser RJ et al. Fluorescence
detection in automated DNA sequence analysis. Nature
1986.321 (6071): 674–9) (Figure 11.1). This method
allows for one reaction to be performed, with all four
ddNTPs in one tube, and was instrumental in the advent
of automated, high volume DNA sequencing.
Figure 11.1. Sequence results with
radioactive (left) and fluorescently
labeled (right) ddNTPs. The
fluorescent results show a distinct
color for each nucleotide (does not
translate well to black and white).
We will be sending your samples to GeneWiz
(http://www.genewiz.com/public/DNA-sequencingservices.aspx). They use Sanger sequencing with
fluorescence to perform high throughput sequencing. To use this service, we must submit a
sample of our plasmid miniprep DNA and either a custom prepared sequencing primer or use a
standard primer which anneals to a portion of the plasmid. The decision of which type of primer
to use will be determined by which mutations you chose and the location within the gene (the
maximum read length is ~1000 bases; you will probably not obtain this maximum).
In order to send the appropriate amount of DNA for sequencing, we will need to quantitate the
DNA. We will be submitting plasmid DNA in the 6-10 kb size range. As seen on the sample
submission guidelines from GeneWiz below, we should be submitting about 800 ng of DNA and
25 pmol of primer in a 15 µL volume. This will require us to quantify the miniprepped DNA.
DNA Type
Plasmids
Template
DNA Length
Concentration
(include vector)
in 10 µl
Template
Total Mass
<6 kb
~50 ng / µl
~500 ng
6 - 10 kb
~80 ng / µl
~800 ng
> 10 kb
~100 ng / µl
~1000 ng
Your
Primer
Total
Picomoles
Premixed Volume*
(Template +
Your Primer)
25 pmol
15 µl
DNA quantitation
Like quantitating the protein yielded by your protein purification, preparations of nucleic acids
are quantitated to determine the relative concentrations of DNA and RNA in the prep and the
relative purity (e.g. presence of contaminants such as protein).
Like proteins, nucleic acids absorb UV light with an optimal absorbance at 260 nm. Beer’s law
and the average extinction coefficient for each nucleic acid can be used to determine the
concentration of the nucleic acid. Sample purity can be assessed by looking at the ratio of
absorbance at 260 nm vs. 280 nm (A260/280). A pure sample of DNA will have an A260/280 ratio
of 1.8, while pure RNA will have an approximate A260/280 ratio of 2.
You will also make note of the A260/A230 ratio. Organic contaminants like phenol, other aromatic
compounds, some reagents used in RNA extraction absorb light of a 230 nm wavelength.
Samples with a low 260/230 (below about 1.8) have a significant presence of these organic
contaminants that may interfere with other downstream processes, including PCR and
sequencing. Absorbance by your miniprepped DNA sample at 230 nm is indicative of
contamination. The 260/230 values for “pure” nucleic acid are often higher than the respective
260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If your sample
is not in this range, you may have problems getting good sequencing results.
Since you have a small DNA sample (40 µL), we will not be using the spectrophotometers in the
lab for quantitation. Instead you will be using a NanoDrop. A NanoDrop is a micro-volume UVVis spectrophotometer which can measure samples as low as 0.5 µL. While the sample is not
recovered, this small volume is not a significant portion of your total volume. The NanoDrop is
attached to a computer and will read at both 260 and 280 nm, will calculate the concentration of
DNA and report the A260/280 ratio. The NanoDrop uses an average extinction coefficient for DNA
to calculate concentration. This instrument is in the Columbus lab in PLSB 131. Your TA will
take select students down to calculate the concentration of your plasmid prep.
II. Required Reading
 Ninfa p. 366-368 (DNA sequencing); 389-396 (PCR)
 Qiagen Qiaprep Spin Miniprep kit product information pdf (collab), p. 9-15, 22-23, and 3942.
 Explore the Genewiz website (http://www.genewiz.com/public/DNA-sequencingservices.aspx) to learn about the process they use
 NanoDrop information pdf (collab) p. 1.1-1.2; 3.1-3.4, 5.1-5.4
III. Pre-lab Assignment
 None
IV. Materials
Qiagen Qiaprep Spin Miniprep kit
Overnight culture of cells with potential
mutant
Centrifuge
Pipettes and pipette tips
Primers in solution
Nanodrop
V. Solutions
Qiagen Qiaprep Spin Miniprep kit solutions provided (buffer P1, buffer P2, buffer PE, buffer N3,
buffer PB)
VI. Procedure
Plasmid miniprep
Night before (to be done by your TA)
1. Pipette 1-5 mL of LB with ampicillin (at 100 µg/mL) (pMH1) or kanamycin (at 50
µg/mL) (pSpeedET) into a culture tube.
2. Inoculate with a single colony from a freshly streaked plate.
3. Incubate at 37 °C with shaking for 12-16 hours (TAs will move the tubes to 4 °C after 16
hours).
During lab:
4.
5.
6.
7.
Transfer 2 mL of culture to each of two 2 mL microcentrifuge tubes.
Harvest bacterial cells by centrifugation in a microcentrifuge at full speed for 3 minutes.
Decant the supernatant to the waste. Invert tubes to allow all media to drain.
Resuspend each cell pellet in 125 µL of Buffer P1 (stored at 4°C). Combine into one
microcentrifuge tube.
8. Add 250 µL Buffer P2; mix by inversion 4-6x. Do NOT vortex. Do NOT mix for >5
minutes. The solution will become cloudy; when you open the tube it will seem thick and
stringy (DNA).
9. Add 350 µL of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6
times. Do not vortex. A white precipitate will form.
10. Spin for 12 minutes at full speed in microcentrifuge. A white pellet will form.
11. During the spin, place a Qiagen column atop a microcentrifuge tube without a cap.
12. Decant or pipette the supernatant from the spin onto the Qiagen column. Do not disrupt
the pellet. IMPORTANT: we are keeping the supernatant at this step; the plasmid is in
the soluble fraction.
13. Spin the column for 1 minute at full speed in the microcentrifuge.
14. Add 500 µL of Buffer PB to the column.
15. Spin for 1 minute at full speed in the microcentrifuge. Discard the flow through (the
DNA is bound to the column). This step eliminates nucleases.
16. Add 750 µL of Buffer PE to wash the column.
17. Spin for 1 minute at full speed in the microcentrifuge. Discard the wash (the DNA is
bound to the column.
18. Spin again for 1 minute to remove residual ethanol from the PE buffer.
19. Move the column to a clean microcentrifuge tube with a cap.
20. Add 40 µL of Buffer EB (Tris/EDTA) buffer or sterile dH2O.
21. Let sit at room temperature for ~1 minute.
22. Spin for 1 minute at full speed in the microcentrifuge.
23. Keep the flow through. This is the eluent with your DNA.
DNA quantitation using the NanoDrop
1. Double click on the desktop NanoDrop software icon and select the Nucleic Acid
application. Follow the prompts for instrument initialization.
2. Pipette 1-2 µL of dH2O on the bottom pedestal. Lower the arm and click the Blank
button. The blank is generally the same buffer that the molecule of interest is dissolved
in.
3. Wipe away the blank with kimwipes.
4. Pipette 1-2 µL of sample onto the pedestal and click Measure.
5. Make notes of the absorbance, concentration, A260/280, A260/230. The software is able to
save information but it is not necessary in this case since you can easily record the values
in your lab notebook.
6. Wipe away the sample with kimwipes.
7. Rinse pedestal by pipetting 1-2 µL of dH2O onto the pedestal; lower the arm; raise the
arm, wipe away the dH2O.
8. Measure next sample, repeating steps 4-7. After the last sample, rinse the pedestal, leave
the arm up, and exit the software.
If your DNA sample is too dilute or impure, consult the troubleshooting guide(p. 36-38) of the
Qiagen product information (on collab). You may have to repeat the miniprep or mutagenesis
protocols, depending on the results of others in your group.
Preparing samples to send for sequencing
Follow the GeneWiz sample submission guidelines:
Template
DNA Length
DNA Type
Concentration
(include vector)
in 10 µl
<6 kb
Plasmids 6 - 10 kb
>10 kb
Template
Total Mass
~50 ng / µl
~500 ng
~80 ng / µl
~800 ng
~100 ng / µl
~1000 ng
Your
Template
Your Primer Primer
Volume
Concentration Volume
Per
µM (pmol/µl)
Per
Reaction
Reaction
10 µl
5 µM
5 µl
1. You will use 0.2 mL PCR tubes that are attached in a strip. They must be carefully
labeled so that each is sequenced properly. There will be 6 samples sent per lab section.
2. Place up to 10 µL of sample in the tube. You want to send about 600-800 ng of DNA.
Ideally you will have roughly 60-80 ng/µL of DNA. If your concentration is much lower,
it is still worth sending for sequencing but the success rate may be lower.
3. If you are using a custom primer, add 5 µL of 5 µM primer. If you are using a standard
primer from GeneWiz (e.g., pBAD forward or reverse), add nothing.
4. Be sure your TA knows what is in each tube and that they are properly labeled.
Interpreting sequence data:
Your sequencing results will be back within 48-72 hours. Your TA will email or post the DNA
sequence for you to analyze.
1. Go to www.expasy.org/translate
2. Paste your DNA sequence into the box.
3. Click on “translate sequence.” You will get back the translated sequence in each of the
three reading frames. The correct reading frame should be easy to identify because there
will be a long stretch of amino acids without a STOP.
4. Find a stretch of amino acids near the residue to be mutated. You can use the find feature
on your computer to find a stretch of amino acids. The sequence should differ at the one
amino acid you chose to mutate if mutagenesis was successful.
5. If your mutagenesis was unsuccessful, check with others in your group to see if theirs
was successful. If none were successful, you will be able to repeat the PIPE mutagenesis
next semester.
VII. POI Report
The POI report (described on p. 179) will include the results of labs 10 and 11 (i.e., was your
mutation successfully introduced and the data that supports this conclusion) and a thorough
discussion of the mutation you chose, the basis for that choice, and the expected result of the
mutation (labs 1B, 5B, and 6). It will also include a discussion of how you will perform the
experiments on your wild-type and mutant POI.