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Cleavage of Vector DNA with Restriction Enzyme(s) and Removal of “Stuffer”
We’ll illustrate processing of vector DNA with fUSE5 (see vectors.doc). This vector has two SfiI sites
separated by a 14-bp “stuffer.” The stuffer is removed by filtration through an ultrafilter with a 100
KDa molecular weight cutoff (Centricon 100 from Amicon). We do not attempt to use calf instestine
phosphatase to remove 5´ phosphates from the cleaved vector. Dephosphorylation may, indeed, be
inefficient with SfiI ends, since they have 3´ overhangs. It’s also probably unnecessary with this vector,
since the two SfiI ends are neither complementary nor self-complementary and therefore cannot be
ligated to themselves or each other.
NOTE: In the document Cleavage of f88.doc we give an alternative protocol, using vector f88-4 as the
illustrative example. In that protocol, isopropanol precipitation is used in place of ultrafiltration
through Centricon 100’s to remove the stuffer.
1. In a 1.5-ml Ep tube pipette
360 g fUSE5 RF
144 l 10 × React 2 buffer
1440 units SfiI
water to bring the total volume to 1.44 ml (add water first)
Vortex gently, microfuge briefly to drive solution to bottom, incubate at 50º 2 hr.
NOTE: See Cleavage of f88.doc for cleavage of the vector f88-4 with HindIII and PstI.
2. Add 72 l 250 mM EDTA to stop digestion; pipette 1/3 of the solution (504 µl) into two additional
1.5-ml Ep tubes.
3. Extract all three tubes once with phenol and once with chloroform, using the double-spin method.
4. Degas the three tubes for ~30 min on the SpeedVac to get rid of most traces of chloroform (the
Centricons to be used in the next step are not resistant to chloroform). We can still smell chloroform
after the degassing, but the Centricons seem to withstand this small amount.
5. Pool all three final aqueous phases in a single graduated tube and add TE to bring the total volume to
4 ml. Add half the solution to each of two Centricon 100’s; concentrate in the SS34 rotor (use thickwalled rubber adaptors) at 2500 rpm at 4º to less than ~100 µl in each Centricon (~1 hr); wash six times
with TE (add TE to the 2-ml fill mark and re-concentrate) to remove the stuffer. This takes 1–2 days to
complete, but of course you can do other things during the long centrifugation steps.
6. After the final concentration step, add 150 µl TE to each of the two retentates; cover with parafilm,
and vortex to ensure mixing of the concentrated DNA solution. Collect the diluted retentates by backcentrifugation into a collection tube as in the Centricon instructions, and pool them in a single tared 1.5ml Ep tube. Add an additional 150 µl TE to each Centricon, vortex, and collect these washes by backcentrifugation; pool these washes with the original retentates in the 1.5-ml Ep tube. Re-weigh the Ep
tube to determine the net weight in grams = volume in ml. The volume of DNA solution should be ~800
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µl. Add additional TE as necessary to bring the total volume to 1.5 ml. The nominal DNA
concentration, assuming 100% yield, is ~240 µg/ml. Store in the refrigerator.
7. Scan 200 l of a 1/30 dilution from 220 to 300 nm. The nominal A260 (assuming 240 µg/ml in the
undiluted stock) will be 0.160, corresponding to 8 µg/ml in the 1/30 dilution. Save the 1/50 dilution for
the next step.
8. In a 500-µl Ep tube mix 20 µl of the 1/30 dilution (~160 ng DNA) and 3.3 µl of 70/75/BPB.
Electrophorese on a 0.8% agarose/4 × GBB gel next to .HindIII or .BstEII markers. Linearized
fUSE5 (the intented product) is 9.2 Kbp long and essentially co-electrophoreses with the 9.4 Kbp
.HindIII band (just behind the 8.45 Kbp .BstEII band); uncut fUSE5 RF electrophoreses as if it were a
linear fragment of ~5 Kbp, while open circular fUSE5 RF electrophoresis as if it were a linear fragment
of ~11 Kbp. Only the linear band should be visible.
NOTE: It should be noted that the check-gel in the previous step does not distinguish molecules that
have been cut at one SfiI site from molecules that have been cut at both sites; and does not test whether
the 14-bp stuffer has been effectively removed. That is why we do the ligation test in steps 9–11. This
test requires a synthetic test insert (see below); if none is available, you’ll have to make due without.
9. In a 1.5-ml Ep tube pipette:
300 ng fUSE5.SfiI (step 6 above)
3.75 µl commercial 10 × ligation buffer (with ATP; don’t use the kind with PEG)
water to bring the total volume to 37.5 µl
Mix and dispense 12.5 µl (1/3 of the volume) to two additional 1.5-ml Ep tubes. There are three tubes in
all, each with 100 ng vector DNA in 12.5 µl 1 × ligation buffer.
10. To one tube add 1 ng of a 20–50 bp synthetic test insert (see below) in 1 µl of TE or other noninterfering buffer; to the other two add 1 µl of blank buffer solution (if possible, the same buffer as the
test insert is in).
11. In a 500-µl Ep tube on ice dilute 1 µl T4 DNA ligase in sufficient 1.09 × ligation buffer to bring the
enzyme concentration to ~10 Weiss units/ml. Add 11.5 µl of this dilution to the tube from the previous
step with the test insert, and to one of the tubes without the test insert. Also, add 11.5 µl of blank 1.09 ×
ligation buffer (no ligase) to the other tube without the test insert. Incubate all three tubes 6–16 hr in a
10º water bath (we simply fill a well-insulated ice-bucket fairly full of 10º water and leave it covered in
the cold room).
NOTE: The synthetic DNA insert must have appropriate ends for ligation to fUSE5. For example, we
made a 33-bp insert by hybridizing an equimolar mixture of two 33-base oligonucleotides with the
sequence
5´-GG|GCG|GAT|TTT|TTG|GAG|AAG|ATT|GGG|GCC|GCT|G-3´
3´-TG|CCC|CGC|CTA|AAA|AAC|CTC|TTC|TAA|CCC|CGG|G-5´
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at 68º at a total DNA concentration (both strands together) of 100–500 µg/ml in 0.5 M NaCl, 50 mM
Tris.HCl pH 8.5 (measured at room temperature). Any other small insert with the appropriate ends
would be O.K.
NOTE: Our test insert has phosphorylated 5´ ends, so that ligation creates covalently closed circular
(but not supercoiled) ligation products. These products consist of ~7 topoisomers, which migrate as
distinct bands on electrophoretic gels in the absence of ethidium bromide and are therefore difficult to
detect. To circumvent this problem, we either use test inserts with 5´ hydroxyls or include 0.5 g/ml
ethidium bromide in the gel (add 1/1000 vol of 0.5 mg/ml stock after melted gel has cooled to 50–60º).
The ethidium induces positive supercoiling in the covalently closed circular ligation products, making
all seven or so topoisomers migrate to about the same position—as if they were ~5 Kbp linear doublestranded fragments in this gel system. In the protocol below, we assume the test insert in not
phosphorylated, so that the intended ligation product is open circular DNA.
12. Add 4.2 l 70/75/BPB to each of the three samples. Run them along with .HindIII or .BstEII
markers on an 0.8% agarose/4 × GBB minigel (add 0.5 µg/ml ethidium bromide if necessary; see note
above). Linear vector (without insert, or with an insert joined only at one end) runs as a 9.2 Kbp linear
double-stranded molecule. Open circular vector (presumably containing an insert) runs like an ~11 Kbp
linear double-stranded molecule. Sometimes slower bands corresponding presumably to higher
multimers are visible. We consider the vector suitable if only linear vector appears in the ligation
without insert and in the mock ligation without ligase; whereas a substantial amount of open circular
vector (at least 10%) appears in the ligation with test insert.