533567836 Page 1 2/16/2016
Preparation of a 6-liter batch of fUSE55 RF using QIAGEN ion exchange chromatography
rather than CsCl/ethidium bromide density equilibrium centrifugation
This protocol serves as an exemplar of our current procedure for preparing large batches of
vector circular double-stranded RF for large library construction. Previously we used CsCl
density equilibrium centrifugation in the presence of a saturating concentration of ethidum
bromide to purify covalently closed circular RF from other contaminants. More recently,
however, we have used ion exchange chromatography on QIAGEN columns for this purpose in
order to avoid working with large amounts of the potentially carcinogenic ethidium bromide.
Although the RF preparation obtained by ion exchange chromatography—the RF “maxiprep”—is
not nearly as pure as that from CsCl/ethidum bromide density equilibrium centrifugation, it
seems to be adequately pure for library construction. The protocol is readily down-sized by a
factor of 6 in obvious ways.
STARTING MATERIALS
fUSE55 midiprep (obtained by ion exchange chromatography on a QIAGEN-tip 100 column);
the nucleic acid concentration measured spectrophotometrically is 851 µg/ml, but the actual RF
concentration as estimated by the intensity of gel electrophoresis bands is probably more like ~72
µg/ml); in this protocol the RF serves simply to transfect electrocompetent cells, so its purity is
not important.
ELECTROPORATION
1. Set up the electroporator at 1250 V and 10 ms (400 ohms), along with following
supplies:





Rack with a 15-ml tube containing 1.5 ml SOC with 0.2 µg/ml tetracycline
Sterile transfer pipette in the 15-ml tube
Space in the S-I for strapping the rack in
Ice bucket with fUSE55 midiprep
A 1-mm cuvette on ice containing 55 µl 20% glycerol underlay (6.9 ml water, 2
ml glycerol, 1 ml TE pH 8, 100 µl 10-µM phenol red)
 Five NZY/Tet plates (containing NZY supplemented with 40 µg/ml tetracycline)
labeled 10-1–10-5
533567836 Page 2 2/16/2016
2. Remove a single tube with a 22-µl aliquot of frozen MC1061 electrocompetent cells
(see ElectrocompetentCells.doc) and immediately do an electroporation as follows
 Thaw the tube of electrocompetent between finger tips
 Pipette 1 µl (851 ng nucleic acid; probably ~72 ng RF) of the fUSE55 midiprep
into the electrocompetent cells and stir with pipette tip; leave on on ice 30 sec.
 Carefully layer 18 µl of the mixture on top of the red 20% glycerol underlay in the
cuvette, being very careful to avoid bubbles.
 Zap
 Using the transfer pipette, draw up the SOC in the 15-ml tube, use it to resuspend
the zapped cells by vigorously pumping up and down a few times, and transfer the
resuspended cells back into the 15-ml tube (don’t worry about the small volume
that remains inaccessible in the cuvette).
3. Shake the 15-ml tube at 37º for 45 min.
4. Meanwhile, label five sterile dilution tubes (capless 2.2-ml tubes; see serial dilutions
in stdpreps.doc) 10-1–10-5 to correspond to the labeled NZY/Tet plates step 7. Pipette 450
µl SOC into all five tubes.
5. When the 45-min incubation step 3 is finished, make five serial 1/10 dilutions of the
electroporation culture in the five dilution tubes by passing 50 µl. Spread 200-µl portions
of all five dilution tubes on the corresponding NZY/Tet plates. Incubate plates overnight
at 37º. Enter the colony counts below.
Dilution
10-1
10-2
10-3
10-4
10-5
Colonies/µg (assuming 72 ng RF)
Colony count
TNTC
TNTC
~1600
186
13
2.6 × 108
NOTE: The estimated cloning efficiency, about 3 × 108 clones per µg of RF, is about 10
times lower than would be obtained from very high quality electrocompetent cells, but
obviously is good enough here since we only need one clone of transfected cells.
PROPAGATION
6. Use a single well-separated colony from the 10-5 plate previous step to inoculate 20 ml of NZY
+ 20 µg/ml tetracycline in a 125-ml culture flask; shake vigorously for several hours; then use
equal 5-ml portions to inoculate six 1-liter cultures of NZY/15 g/ml tetracycline in 2.8-liter
Fernbach flasks. Label the cultures 1–6 and keep track of culture numbers. Shake vigorously
overnight (~16 hr).
533567836 Page 3 2/16/2016
NOTE: In preparation for step 11 next day, inoculate ~3 ml NZY in a 1 × 3.5 inch culture tube
with 30 µl of a K91BK (see Strains.doc) glycerol tube.
7. Use each culture to fill two labeled 500-ml centrifuge bottles all the way to the neck (12
bottles altogether); just leave the leftover culture in the flask—it’s not worth saving.
8. Remove 200 µl from one of the bottles from each culture to a sterile labeled 500-µl Ep tube
(six Ep tubes altogether); centrifuge briefly; carefully transfer 100 µl of each supernatant to a
fresh sterile labeled 500-µl Ep tube. Store temporarily in the refrigerator while going on to next
two steps.
9. Screw the caps on tightly onto the 500-ml centrifuge bottles; keep the bottles cold during the
remainder of this step. Centrifuge bottles 5 Krpm 15 min in GS3 rotor at 4º; RRR.
10. Add 75 ml 50 mM EDTA (need not be sterile) to each 500-ml bottle and shake to thoroughly
suspend the cells. Combine the cells from both bottles for one culture in a single high-speed
Nagle 250-ml centrifuge bottle (need not be sterile). Centrifuge at 5 Krpm at 4º for 10 min in the
SLA1500 rotor, using the white cushions; RRR; freeze cell pellets at -20C.
NOTE: The fUSE55 vector, like fUSE1, fUSE3 and fUSE5, is non-infective as a result of a
frameshift mutation in gene III (see vectors.doc). The next two steps are intended to confirm that
very few of the fUSE55 clones have acquired mutations that make the particles infective (see
fUSE135propagation.doc); such mutants would be pseudorevertants, because it’s extremely
unlikely that any random mutation process would restore the original wild-type gene-III
sequence. Pseudorevertants can’t be avoided altogether, but an infective titer less than about 106
TU/ml (about 0.004% of the wild-type titer) is perfectly acceptable.
11. Make a 20-ml batch of K91BK starved cells as usual (see TUtiter.doc). (NOTE: If
convenient, this can be done the day before next step.)
12. Dilute 10 µl of the supernatants step 8 (500-µl Ep tubes) into 990 µl TBS/gelatin in a sterile
dilution tube. Titer 10-µl portions of the 1/100 dilutions on K91BK starved cells as usual (see
TUtiter.doc; use plates containing NZY supplemented with 40 µg/ml tetracycline and 100 µg/ml
kanamycin), along with 10 µl TBS/gelatin diluent as negative control and 10 µl of a positive titer
control containing 1.8 × 106 virions/ml in TBS/gelatin. Typical results are shown below:
Sample
Dilution
fd-tet positive control
10-7
Culture 1
10-2
Culture 2
10-2
Culture 3
10-2
Culture 4
10-2
Culture 5
10-2
Culture 6
10-2
TBS/gel negative control
Colonies
272
5
1
2
3
2
4
0
Titer (TU/ml) Virions/ml
1.39×1012
1.816×1013
5
2.6×10
5.1×104
1.0×105
1.5×105
1.0×105
2.0×105
Infectivity
7.64%
533567836 Page 4 2/16/2016
Since the titer of of infective pseudorevertants is acceptably low (<106 TU/ml), we proceeded
with RF isolation with all six cultures.
NOTE: Do steps 13–22 below separately for each 1-liter culture. The six preps won’t be pooled
until after the check gel step 23.
13. Thaw and suspend each frozen cell pellet step 4 (high-speed 250-ml bottles in deep freeze)
in 40 ml buffered glucose by vigorous shaking or vortexing.
14. Add 80 ml 0.2 N NaOH/1% SDS (freshly made from 2 N NaOH stock). Mix by gentle
inversion, put on ice 15 min.
15. Add 60 ml cold “5 M” KOAc; mix by gentle inversion.
16. Centrifuge at 12 Krpm 15 min at 4º in the SLA1500 rotor. There will be a gelatinous pellet
at the bottom of the bottle, and a stringy, gelatinous precipitate throughout the supernatant. Don't
worry—press on!
17. Pour the supernatant through 2-3 layers of cheesecloth into a beaker (this gets rid of most of
the stringy, gelatinous precipitate), and divide the filtered supernatant (still pretty cloudy) equally
into four 50-ml screw-cap centrifuge tubes. Centrifuge at 14 Krpm 15 min at 4 in the FiberLite
F15S-8×50C rotor1 (you’ll need three runs on the rotor to centrifuge all 6 × 4 = 24 50-ml tubes).
The supernatant should be clear; often it has a slight greenish tinge.
18. Pour all four supernatants (from one 1-liter culture) into a single tared 250-ml centrifuge
bottle (needn’t be the high-speed kind), determine net weight, pour 1/3 of the supernatant into
two more 250-ml centrifuge bottles. (You’ll need 18 of these bottles altogether.)
19. To each bottle previous step add 150 ml 95% ethanol; allow to sit in cold at least 30 min;
the DNA can be stored this way overnight or several days if convenient.
20. Centrifuge bottles at 8 Krpm 20 min in SLA1500 rotor; RRR; wash pellets with 20 ml 70%
ethanol (made from 95%; need not be cold); RRR; dry briefly under vacuum. (This step will
have to be done three times altogether to process all 18 bottles.)
21. Dissolve and pool the pellets from all three bottles from a single culture in a total volume of
10 ml TE; transfer to a single 50-ml screw-cap centrifuge tube.
22. Centrifuge at 14 Krpm 5 min in FiberLite rotor; pour each supernatant into a 15-ml tube;
store in refrigerator while awaiting the results of the next step.
1
These excellent rotors allow you to spin standard disposable 50-ml conical screw-cap centrifuge tubes at
15,000 rpm! The pellet collects at the angle rather than the tip, which makes it particularly easy to free of
residual supernatant. If you don’t have this rotor, you can use OakRidge tubes and the Sorvall SS34 rotor
(or equivalent Beckman rotor) instead.
533567836 Page 5 2/16/2016
23. Run 1 µl of the six preps (each diluted with 7 µl water and 2 µl lysis mix) on a 0.7%
agarose/4×GBB minigel next to 10 µl 40 µg/ml .BstEII marker. Stain with SybrGreen and
photodocument as usual. Here are typical results:
NOTE: The most prominent band, co-migrating with the 4.8-Kbp linear double-stranded
standard band, is covalently closed circular RF; the diffuse band co-migrating with a ~2.5-Kbp
linear double-stranded fragment, is probably single-stranded viral DNA; the sharp but fairly weak
band between the 8.45- and 14-Kbp markers is open circular RF; the extremely intense, diffuse
blob near the bottom of each lane is RNA. The above pattern indicates excellent crude RF
preparation.
24. Pool all six preps in a single 125-ml Nalge bottle (total volume ~60 ml); divide the solution
evenly into four 50-ml screw-cap tubes (should be ~15 ml each). Phenol extract as follows:





Shake each tube vigorously with an equal volume of freshly neutralized phenol
Centrifuge 5 min at top speed in the clinical centrifuge to separate phases
Transfer each aqueous (upper) phase GREEDILY into a 15-ml screw-cap centrifuge tube
Centrifuge the 15-ml tubes 5 min top speed in the clinical centrifuge
Transfer each aqueous phase CAREFULLY to a fresh 50-ml screw-cap tube
Repeat the phenol extraction and do a chloroform extraction using the same method, except pool
all four final aqueous phases in a single 125-ml Nagle bottle, then divide the pooled aqeous
phase evenly into two 50-ml screw-cap tubes (nominally 30 ml each).
533567836 Page 6 2/16/2016
25. To each screw-cap tube add ½ vol 7.5 M NH4OAc; leave on ice 15 min.
26. Centrifuge at 14 Krpm 15 min in FiberLite rotor; this pellets large RNA species. Pour the
cleared supernatants into a single 125-ml Nagle bottle and divide the supernatant solution equally
into eight 50-ml screw-cap tubes.
27. To each tube add 2.35 vol (theoretically 26.5 ml, but in practice probably less) 95% ethanol.
Allow to precipitate at least 15 min on ice (or refrigerator overnight).
28. Centrifuge 14 Krpm 15 min in FiberLite rotor; RRR; air-dry; dissolve each of the eight
pellets in 1 ml TE and pool them in a single one of the 50-ml tubes (total volume theoretically 8
ml).
29. Into the 50-ml tube pipette 8 µl 10-mg/ml RNaseA; vortex gently; centrifuge briefly
in the clinical centrifuge; incubate at 37º 15 min.
NOTE: RNaseA digestion is delayed until after the ammonium acetate precipitation step
25, which is reported to pellet large RNA species (e.g., ribosomal RNAs). Those large
RNAs would probably contain folded sub-structures that are resistant to RNaseA
digestion but too small to precipitate in concentrated ammonium acetate.
30. Centrifuge briefly in clinical centrifuge; transfer half the solution (4 ml) to a second
50-ml tube; to each tube add 36 ml ml QIAGEN buffer QBT; vortex thoroughly to mix;
centrifuge the tubes briefly in the clinical centrifuge to drive the solutions to the bottom.
NOTE: At this stage the crude RF DNA is sufficiently pure to be chromatographed on six
QIAGEN-tip 500 maxiprep columns. A standard alkaline lysate prepared as
recommended in the QIAGEN manual for high copy number plasmids would
undoubtedly be far beyond the capacity of six columns.
31. Mount six QIAGEN-tip 500s in a suitable rack over a waste container. Equilibrate
each tip with 10 ml QIAGEN buffer QBT, allowing the effluent to drain to the waste.
The columns will stop flowing automatically when the meniscus reaches the upper frit.
32. Still collecting to waste, apply 13.3 ml of step 30 (six columns should use up the
solution) to each column and allow the entire sample to flow through.
33. Still collecting to waste, wash each column twice with 30 ml QIAGEN buffer QC,
allowing the entire wash buffer to drain through each time.
34. Position each QIAGEN-tip column to collect into a fresh 50-ml tube; elute the
column with 24 ml QIAGEN buffer QF; again, allow the buffer to flow completely
through the column.
533567836 Page 7 2/16/2016
35. To each 50-ml tube (with 24 ml eluate) add 17 ml isopropanol (total volume should
now be 41 ml); mix thoroughly by vortexing; mark one edge of each tube to designate the
centrifugal wall (where the pellet will end up); orienting the marked edges outward
(centrifugally), centrifuge in two runs at 14 Krpm at 4º for 30 min in FiberLite rotor; RRR
(be very careful and gentle as glassy pellet may not be visible); air-dry briefly (no need to
get rid of last drops of liquid).
36. Dissolve each pellet in 1.9 ml TE by soaking overnight with occasional vortexing;
centrifuge the 50-ml tubes to drive the solution to the bottom; pool all the dissolved
pellets in a single one of the 50-ml tubes; dialyze the pooled solutions in a single 12-ml,
10-KDa-cutoff Slide-A-Lyzer against three changes of 900 ml of 1/10 × TE; save some of
the last outside fluid to use as diluent and reference at step 38.
37. Remove dialysate from the Slide-A-Lyzer into a tared 15-ml bottle or other suitable
container, recording the net weight = volume in the table next step; store the RF maxiprep
in the refrigerator or freezer.
38. In a 500-µl Ep tube make 100 µl of 1/5 dilution of the maxiprep and scan from 220–
320 nm, using the saved final outside dialyis fluid step 36 as diluent and reference. Data
are graphed and analyzed below:
0.6
Absorbance
0.5
0.4
Dialysis Buffer TE 1:10
Remove Blank Rescan
Remove Blank Rescan
fUSE55RF2step39 1:5
0.3
0.2
0.1
0
-0.1
220
240
260
280
300
320
Wavelength
Corrected net A256
Undiluted nucleic acid concentration
Volume (previous step)
Total yield from 6 liters
Yield from each liter
0.584
146 µg/ml
16.366 ml
2390 µg
398 µg
There are evidently lots of contaminants that absorb in the far UV; that is probably the reason
why the peak absorption occurs at 256 nm rather than ~258 (however, we don’t attempt to
533567836 Page 8 2/16/2016
compensate for this error). It’s probable that a substantial amount of the nucleic acid in this
QIAGEN-purified RF is actually not RF, but the preparation seems to be serviceable for library
construction anyway.