Amplification and storage-
- Remove GIPz clones from -80°C storage and inoculate 2 µl primary stock culture per 2 ml LB containing
25 µg zeocin, 100 µg/ml carbenicillin and 8% glycerol
- Incubate with shaking at 30°C for 16-20
hours.
- Freeze at -80°C for
long-term storage.
Plasmid Prep-
(A 5 ml culture of shRNAmir construct
is sufficient to produce 5-10 µg of plasmid
DNA.)
- Thaw a GIPz glycerol stock
and pulse vortex to suspend the E. coli.
- Transfer a 10 µl inoculum of the glycerol stock to 5 ml of low-salt LB containing 100 ?g/ml carbenicillin.
(Return glycerol stock to -80°C.)
- Incubate at 37°C for 16 hours with vigorous shaking. (If larger cultures are needed, use
this 16-hour culture as inoculum for a larger culture.)
- Centrifuge the culture, and
recover the bacterial pellet for plasmid DNA preparation.
- Run 200 ng (generally 3-5 µl) of the plasmid DNA on a 1% agarose gel. (The pGIPz + shRNAmir is 11,744 bp.)
- To avoid formation of recombinants,
keep all incubation times as short as possible, and avoid subculturing.
- The bacteria can be grown
at 37°C
for template prep or sequencing. However, for archival replication
grow all pGIPZ clones at 30°C in
low-salt LB with zeocin and carbenicillin.
Restriction Digest of GIPZ-
- Using filter pipette tips
and sterile conditions make the following additions to a sterile PCR
thin-wall tube:
- Sterile nuclease-free water x µl
- Restriction enzyme buffer,
10-x 1 µl
- BSA (10 mg/ml stock) 1 µl
- DNA sample (80-240 ng in d.w.
or TE) x µl
- Restriction enzyme, 20 U 0.25 µl
- Final volume 10 µl
- Mix gently by pipetting.
- Incubate in thermal cycler
at 37°C
for 2 hours.
- Load 10 µl of
the digested sample on a 1% agarose gel.
Transfection-
(to evaluate the quality of the clones)
Protocol assumes transfection of shRNAmir
plasmid DNA into HEK293T cells in 24-well clusters using medium that
does not contain serum or antibiotics.
- Warm the Arrest-In transfection
reagent to ambient temperature (~20 minutes on the bench) and mix by
vortexing or inversion prior to use.
- Maintain sterile conditions
working with the DNA and Arrest-In as the mixtures will be added to
cells.
- One day before transfection
plate the HEK293 cells at 5E4/well in a 24-well cluster using complete
medium with serum and antibiotics.
- On the day of transfection
prepare the DNA/Arrest-In complexes.
- Set up two microfuge tubes
for each well to be transfected:
- Dilute 500 ng shRNAmir into
a final volume of 50 µl serum-free
medium.
- Add 2.5 µg
(2.5 µl) of Arrest-In to 47.5 µl serum-free medium.
- Transfer the diluted DNA from
tube 1 to the diluted Arrest-In (tube 2); mix rapidly, and incubate
for 20 minutes at room temperature. (Note: 1:5 DNA:Arrest-In ratio.)
| Transfection |
shRNAmir plasmid |
Reporter |
Carrier DNA |
| Gene of interest |
500 ng hairpin to target |
0 |
0 |
| Transfect. efficiency |
0 |
500 ng |
0 |
| Knockdown efficiency |
450-500 ng hairpin to reporter |
50 ng |
0 |
| Knockdown control |
0 |
50 ng |
450-500 ng |
| Non-silencing control |
500 ng control hairpin |
0 |
0 |
Separate reporter transfection is not
required when construct already contains a reporter (e.g. GFP, luciferase
and/or β-gal.)
Use carrier-DNA such as pUC19 or pBluescript plasmid as needed to maintain
optimal DNA/Arrest-In ratios.
- Add an additional 150 µl
serum-free
medium to each of the tubes containing the DNA/Arrest-In complexes,
and mix gently. Aspirate the growth medium from the HEK293 cells,
and add the 250 µl
DNA/Arrest-In complexes. Incubate for 3-6 hours in a CO2
incubator at 37°C.
- After the initial incubation
add 250 µl
additional growth medium containing 2X serum (e.g. 20 % serum) and return
cells to the incubator. (If toxicity of the transfection complexes
is a problem, remove the medium from the cells after the initial incubation,
and replenish with 500 µl
medium with 10% FBS.)
- Observe cells 48-96 h after
transfection for evidence of reporter expression. Then evaluate
cells for reduction in gene/reporter by qRT-PCR, or western blot.
(Optimal duration of transfection-to-analysis
will depend on cell type, gene of interest, stability of the mRNA and/or
protein.)
- When selection of stably transfected
cells is desired transfer the cells to medium containing puromycin,
but this selection process should be delayed at least 48 hours.
The puromycin working concentration varies from 1-10 µg/ml
depending on the cell line. The lowest
concentration should be used that can be shown to kill 100% un-transfected
cells within 1-4 days.
(Optimum conditions for transfection
can be determined by varying DNA:Arrest-In ratio, DNA concentration
and cell confluency.
Packaging Lentivirus-
Use the Trans-Lentiviral shRNA packaging
System (TLP4614 or-15) to generate a replication-incompetent HIV-1-based
lentivirus which may be used to deliver and express the gene of interest
or shRNAmir in either dividing or non-deviding mammalian cells.
The kit provides sufficient packaging
mix and Arrest-In for 10 packaging events in 100 mm plates.
Kit contents:
- Trans-Lentiviral packaging
mix containing optimized mixture of five packaging plasmids (pTLA1-Pak,
pTLA1-Enz, pTLA1-Env, pTLA1-Rev and pTLA1-TOFF).
- TLA-HEK293T cells for production
of lentivirus following co-transfection of the transfer plasmid and
the packaging plasmids. The TLA cell line stably expresses SV40
large T antigen and facilitates production of high viral titers.
- Arrest-In proprietary lipo-polymeric
transfection reagent which optimizes uptake and transfection of shRNA
plasmid DNA into the nucleus of eukaryotic cells
- pGIPZ non-silencing control
vector for use as expression control to generate non-silencing lentiviral
stock to optimize expression conditions in the mammalian cell line of
interest.
- pGIPZ cloning vector containing
Turbo GFP reporter and also elements required to allow packaging of
the expression construct into virions (i.e. 5' and 3' LTRs and Ψ packaging
signal.
Transfection and Virus production-
(optimized for transfection of shRNA
plasmid DNA into TLA-HEK293T cells in 100 mm tissue culture dishes using
medium without serum or antibiotics.)
- One day before transfection
plate the TLA-HEK293T cells at 5.5E6/100mm dish in 10 ml complete medium
with serum and antibiotics. This provides an initial cell density
of 1E5/cm2.
- On the day of transfection
prepare DNA and Arrest-In as described above except that for each plate,
dilute 37.5 µg
DNA (26 µl
packaging mix containint 28.5 µg DNA and
9 µg pGIPZ transfer vector) into 1 ml
final volume
serum free medium (in a microfuge tube), and dilute 187.5 µl Arrest-In
(1 mg/ml stock) up to 1 ml in serum-free medium (in a 5 ml polypropylene
tube).
- Transfer the diluted DNA to
the tube containing the diluted Arrest-In, mix rapidly, and then incubate
for 20 minutes at room temperature.
- Add an additional 3 ml of
serum-free medium to each of the tubes containing the DNA/Arrest-In
complexes, mix gently, remove the growth medium from the cells, and
add the complexes to the cells. Return the cells to the CO2
incubator at 37°C
for 3-6 hours.
- After the 3-6 hour exposure
of the HEK cells to complexes gently aspirate the transfection mixture
and replace with 12 ml standard culture medium.
- After 48-72 hours examine
the cells microscopically for TurboGFP expression.
- Harvest the virus-containing
supernatants 48-72 post-transfection by removing the medium for storage
in a 15 ml, sterile conical tube.
If the virus is to be concentrated, harvest
the supernatant at two intervals, 48 and 72 hours after transfection.
At 48 hours, collect the supernatant in a sterile 50 ml conical tube
for storage at 4°C.
Add 12 ml complete medium to the transfected cells, and after an additional
24 hours incubation, harvest the culture supernatant, and pool with
the 48-hour supernatant.
Caution: The viral particles
in the supernatant are infectious. Use recommended procedures
for BL-2 organisms.
- Centrifuge the harvested supernatant
3000 rpm, 20 min, 4°C to pellet cell
debris.
To concentrate the virus centrifuge
at 23,000 rpm using SW28 centrifuge tubes for 1.5 hours at 4°C.
Aspirate and discard the supernatant. Resuspend the pellet in
DMEM (without serum), using ~100 µl volume
per pellet derived from one 100 mm plate
or ~250 µl
for a pellet derived from one SW28 tube containing 35 ml supernatant.
The pellet is comprised mostly of serum proteins. After adding
the medium for resuspension, let stand for 5-10 minutes followed
by gentle pipetting ~30 times. Avoid bubbles. Transfer the
resuspended protein/virus to a microfuge tube and spin full speed for
3-4 minutes to pellet most of the serum proteins. Transfer the
virus-containing supernatant to a fresh tube; aliquot into multiple
tubes for storage at -80°C.
Viral stocks should be stable for
~ 1 year at -80°C.
Store as multiple aliquots to avoid freeze/thaw cycles which result
in loss of viral titer.
Determine the titer of the silencing
lentiviral stock as well as non-silencing controls. This may be
done in TLR-HEK293T cells or preferably in the cells that will be used
in experiments.
- One day before transduction
seed a 24-well cluster with target cells (e.g. TLA-HEK293T cells) at
5E4 cells/well in DMEM containing 10% FBS and 1% Pen/Strep. On
the following day the cells should not be more than 40-50% confluent.
- Prepare dilutions of the viral
stock using a round bottom 96-well plate using dilution medium (DMEM
containing 0.5% FBS and 8 mg/ml
polybrene. (The polybrene is a
cation which coats the viral particles, conferring a net positive charge
to counteract the negatively-charged cell surface.)
- Add 80µl
of the dilution medium to columns 1-8 of as
many rows as needed (using one row to titrate each viral stock).
- Add 20 µl
of each thawed virus stock to the designated
well in column 1 (1/5 dilution). Pipet up and down 10-15 times,
and discard tip.
- Using a fresh tip(s), transfer
20 µl
from the well(s) in column 1 to the corresponding well in column 2.
Pipet 10-15 times and discard tip(s).
- Continue the serial dilution(s)
from the well(s) in column two through column 8, yielding a final dilution
of ~1.390K.
- Allow the freshly prepared
virus dilutions to stand at room temperature for 5 minutes.
- Label the 24-well cluster
containing the mammalian target cells, marking one row for each viral
preparation to be tested.
- Remove the culture medium
from the 24-well plate, and add back 150 µl of
transduction medium (DMDM + 0.5% FCS but
without polybrene).
- Transduce the cells by adding
25 µl
of diluted virus from the 96-well plate, e.g. using dilutions from columns
2-7, 1-6 or 3-8 depending on the estimated viral titer.
- Incubate the transduced cultures
4 hours at 37°C
in a CO2 incubator.
- Remove the transduction supernatant
from the cultures, add 1 ml DMEM containing 10% FBS and 1% pen/strep,
and return to incubator for 48 hours.
- Using the fluorescence microscope,
count the numbers of TurboGFP expressing cells (or colonies of cells,
as proliferation should have occurred).
- The calculated viral titer,
TU/ml = Number of GFP-positive cell/colonies x dilution x 40.
When the titer of the viral preparation
is determined, the optimal MOI will need to be established for the mammalian
cell line of choice. The optimal MOI will depend on several variables,
including the nature of the mammalian cell line (e.g. dividing or non-dividing)
its transduction efficiency, the gene of interest, etc. Initial
experiments should be carried out using a range of MOIs (e.g. 0, 0.5,
1, 2, 5, 10, 20 etc). To achieve single copy knockdown an MOI
of 0.3 should result in fewer than 4% of the cells receiving more than
one insert.
- Plate 5-8E4 cells per well
in 24-well plates, and incubate overnight at 37°C
in a CO2 incubator.
- Prepare medium containing
various concentrations of puromycin over the range 0-15 µg/ml, and
replace the growth medium in the 24-well cluster with the medium containing
various concentrations of puromycin.
- Return the clusters to the
incubator, and every 2-3 days replace the culture medium with freshly-prepared
selective medium.
- Monitor the cells daily, and
count the numbers of surviving cells.
- The minimum antibiotic concentration
to use is the lowest concentration that kills 100% of the cells in 1-4
days from the start of antibiotic selection.
- Plate cells, and incubate
overnight.
- e.g. 5-8E4 293 cells/well
in 24-well cluster
- 3-5E5 MEF per 9.5cm2
well (6-well cluster)
| Area and Nominal Culture Volumes of Dishes and Clusters |
| 100mm (style) dish |
55 cm sq. |
5 - 12 ml |
| 24-well cluster |
1.9 cm sq. |
0.3 - 1 ml |
| 48-well cluster |
0.95 cm sq. |
0.15 - 0.5 ml |
| 96-well cluster |
0.32 cm sq. |
50 - 100 µl |
- After the overnight incubation
remove the medium and add the virus to the optimum MOI in a sufficient
volume of serum-free medium to cover the cells.
- This may be a small volume
of concentrated virus + fresh serum-free DMEM or a larger volume of
viral-containing unconcentrated supernatant from HEK293 cells.
- Typical final volume would
be ~175-200 µl
in a 24-well cluster. Use 1 ml medium per well in a 6-well cluster.
- Transduction efficiency may
be improved substantially by addition of Polybrene. The Polybrene
may be added directly to the virus supernatant before mixing with transduction
reagent, or the Polybrene may be added to the cell culture immediately
after addition of virus. Usual final concentrations are 4-10 µg/ml.
- When cells have been shown
to resist viral transduction, centrifugation may improve efficiency.
After addition of virus and polybrene to the cluster, place the cluster
in a centrifuge carrier and spin in the RT600/H-1000B rotor. BMDM
as well as MEF cultures have tolerated 600-1000 x g for 30 minutes at
20°C.
- When virus-containing supernatant
is yellow/acidic/metabolically depleted, addition of fresh medium may
be helpful. For example, 6-8 hours after transduction add an additional
1 ml of complete medium (DMEM + 10% serum and pen/strep) and return
the clusters to the incubator.
- At 48 hours post transduction,
replace the growth medium with complete medium containing puromycin
(as indicated in the above kill curve), and return the cluster to the
incubator.
- Replace the medium every 2-3
days with freshly made selective medium.
- Monitor the cells daily to
observe percentage of surviving cells as well as the level and percentage
of GFP positive cells. Eventually all of the surviving cells should
be GFP positive on or around day three of selection.
The puromycin concentration should
be chosen relative to the MOI used for transduction. When higher
MOIs are used more copies of the shRNAmir and puromycin resistance gene
will be expressed per cell, and higher concentrations of puromycin will
be tolerated by the cell culture. Accordingly, the puromycin concentration
can be adjusted to select for the desired population, though the puromycin
concentration should always exceed the minimum concentration indicated
in the kill curve.
Caveats:
- Generic HEK293 cells can be
used for packaging, but the TLA-HEK293T cell line from Open Biosystems
(which stably expresses the SV40 large T antigen) should provide higher
virus titers. The Invitrogen HEK293FT cells are comparable but
will need to be grown in the presence of Geneticin (G418).
- Always use purified plasmid
DNA, and determine the concentration by OD260.
- Always carefully mix the transfection
reagent and also the DNA/Arrest-In complexes before addition to cells.
- Transfection cytotoxicity
may be minimized by optimizing the DNA:Arrest-In ratio.
- Perform transfection in serum/antibiotic-free
conditions unless cell viability requires serum.
- High protein expression levels
can cause cytotoxicity.
- After transfection with the
transfer plasmid and packaging mix the HEK293 cells will began to express
VSV-G glycoprotein which causes the cells to fuse, forming multinucleated
syncitia.
- Use cells that have a known/documented
history regarding passage number and growth density. In general
better results will be obtained with low-passage-number cells.
Cells should be freshly plated at appropriate density on the day before
transfection.
Evaluation of Knockdown
- Extract cellular RNA (TRIzol
Plus RNA Purification kit, Invitrogen)
This procedure uses TRIzol reagent
to lyse cells, addition of chloroform to obtain an aqueous phase that
contains RNA and a silica spin column to bind and elute RNA. Note:
Wear gloves, lab coat and eye protection. The TRIzol contains
phenol and guanidine isothiocyanate, and the samples should be handled
in the Plexiglas hood until the RNA-containing aqueous phase is transferred
to an RNA spin cartridge.
- Lyse adherent cells by adding
1 ml TRIzol /per 10 cm2 (i.e. 6 well cluster). Use
200 µl for 24-well plate and 100 µl for 48-well plate. When
treating cell suspensions, spin and discard culture medium, and add
1 ml TRIzol per 5-10 E6 cells. In either case, lyse cells by pipetting
up and down. Transfer cell lysate to an Eppendorf tube, and stand
5 minutes at room temperature.
- Add 0.2 ml chloroform per
1 ml TRIzol reagent; cap securely and shake vigorously by hand for 15
seconds. (Too vigorous shaking, e.g. vortexing, may introduce
DNA contamination into the RNA.) Stand 2-3 minutes at room
temperature.
- Centrifuge 12K x g for 15
minutes at 4°C. (The mixture should separate into a lower red
phenol-chloroform layer and a colorless upper aqueous layer containing
RNA, ~600 µl... or 50% of the original 1.2 ml).
- Transfer the top (RNA) layer
to a fresh tube and add an equal volume of 70% ethanol to obtain a final
concentration of 35%; mix well by vortexing. Continue to mix as
required to disperse any visible precipitate.
- Transfer the RNA to a spin
cartridge (up to 700 µl) and spin 12K x g for 15 seconds at room temperature.
Discard the flow-through.
- Add 700 µl wash buffer I
to the RNA spin cartridge and spin 12K x g for 15 seconds at room temperature.
Discard the collection tube.
- Place the RNA spin cartridge
in a fresh wash tube, and add 500 µl wash buffer II (containing ethanol),
and spin 12K x g for 15 seconds at room temperature. Discard the
flow-through.
- Centrifuge the empty RNA spin
cartridge 12K x g for 1 minute at room temperature to remove any remaining
liquid.
- Add 500 µl wash buffer II
and spin 12K x g for 15 seconds; discard flow-through, and then spin
empty cartridge 12K x g for 1 minute at room temperature.
- Place the RNA spin cartridge
in a clean RNA recovery tube.
- Add 30-100 µl RNase-free
water to the center of the cartridge membrane, and stand at room temperature
for 1 minute.
- Centrifuge the spin cartridge
for 2 minutes at 12K x g at room temperature. Discard the spin
cartridge.
- Determine the concentration
and quality of the recovered RNA (OD 260/280) and store at -20°C.
RNA message levels are determined
by qRT-PCR using commercially available reagent kits and supplies.
Two Applied Biosystems 7900 Fast Real-Time PCR systems are used configured
with the standard or the fast sample block respectively. In either
case the 96-well reaction plates are sealed with optical adhesive covers
and loaded into the cycler using a standard compression pad. Generally
one-step kits are used, e.g. AB TaqMan RNA-to-CT 1-Step,
or Invitrogen SuperScript III Platinum One-Step Quantitative RT-PCR
System, using either TaqMan Gene Expression Assay components or LUX
Assays (Light Upon eXtension primers, Invitrogen). Where appropriate,
one-step SYBR green reagents are used with unlabeled primers.
All assays contain ROX reference dye (where possible), and relative
quantitation of target genes vs reference (calibrator) gene is determined
(ΔΔCT)
- Prepare Cell Lysates for
Western Blot Analysis
Cell Lines,
Media and Reagents:
DMEM
(high glucose)
10%
FBS (unheated)
1%
L-Glutamine/Pen/Strep 100x stock
Notes:
- The TLA-HEK293 cells do not need to be maintained in selection medium.
- Transfection should be performed using medium without serum or antibiotics.
- Working without antibiotics is a good idea and can be successfully done without infection using careful
technique.
Starting
cells from frozen stock-
- Remove cells from liquid nitrogen
storage and quick-thaw in 37°C water,
removing the vial just as the last ice
crystals melt.
- Immediately dilute the thawed
cells into cDMEM (with 10% FBS and L-glutamine) in a 15 ml conical tube.
- Spin 1000 rpm for 3-5 minutes
at room temperature (~20°C)
- Carefully pour off the supernatant
medium and resuspend the cells in 15 ml cDMDM, and transfer to a T25
flask or 100 mm dish.
- Place in the CO2
incubator at 37°C.
- Fresh medium should be added
every three days as indicated by medium color. Gentle treatment
is required as the cells are easily dislodged from the flask surface.
- The cells are passaged when
90% confluent and can be split using a 1:15 to 1:20 ratio. This
will permit the cells to go for a number of days before reaching confluency,
thus minimizing the amount of work required to maintain the cell line.
Smaller passage ratios (e.g. 1:5) should be used if large numbers of
cells are needed within a few days.
- Carefully remove the growth
medium (by aspiration) followed by one wash with DPBS to remove medium
components
- Add 0.05% trypsin solution
(Gibco 25300) 2-4 ml per T150 flask (or proportional t the growth surface
area of the flask or dish). Rock the flask gently to coat the
surface, and then aspirate the excess, leaving a thin film of trypsin
on the cells. The flask may be returned to the incubator for ~2
minutes to detach the cells.
- Add cDMDM to the flask using
a 10 ml pipet, and pipet up and down ~5 times to produce a single cell
suspension.
- Dilute the cell suspension
into flasks or dishes as needed, and return to the CO2 incubator.
Microbiological Reagents
Note: Open Biosystems recommens
low-salt/rich medium for production of plasmid, while Qiagen recommends
standard LB (not enriched) broth with high salt (10 g NaCl/liter) for
optimum plasmid production. However, because Zeocin is salt-sensitive,
Invitrogen recommends selective growth in low salt medium (5 g NaCl/liter).
Therefore, use the Luri Bertini Lennox, unsupplemented as shown below
(also checking pH to ensure Zeocin stability).
2X-LB Broth (high nutrient,
low salt, from Open Biosystems handbook)-
Note: This medium is modified from
LB Broth Base (LB Broth, Lennox). The InVitrogen product #12780-052
contains 10g Select Peptone 140 and 5g Select Yeast Extract plut 5g
NaCl per liter.
The above medium can be formulated
using separate components; or, the Invitrogen LB Broth base can be used
(20g/liter, which contains 10g peptone, 5g yeast extract and 5g NaCl)
by supplementation with an additional 10g peptone (Fisher Tryptone BP1421-500)
and 5g yeast extract (EMD 1.03753.0500).
Microwave until just starting to
boil; then autoclave 121°C, 15 psi, 15
minutes.
When cool, add either sterile d.i.
water or 50% glycerol 80ml to 420ml 2X-LB, 16ml to 84ml, 8 ml to 42
ml, etc as needed. The glycerol is for preparation of frozen stocks
while the 2X-LB broth without glycerol is for plasmid preparation, etc.
Antibiotic Stocks-
Zeocin
(Invitrogen R250-01) sterile 100 mg/ml stock is stored at -20°C.
Protect from light, and thaw on ice. Freshly autoclaved media should
be cooled to 55°C
or lower, and Zeocin added to final concentration of 25 µg/ml
for
selection in bacterial cultures (e.g. 12.5 µl
per 50 ml medium). Because Zeocin is
salt and pH sensitive, the recommended growth medium is low-salt LB
(e.g. Invitrogen LB broth base, Lennox, 12780-052, which contains 10g
Tryptone, 5g Yeast extract and 5 g NaCl per liter). Weigh the
powder (20g/liter) as needed, check the pH, and adjust to pH 7.5.
When plates are needed, add 15g/liter of agar (Bacto 214010) microwave
until agar is melted and dissolved (just beginning to boil). Autoclave
20 minutes at 15 psi, 121°C.
Carbenicillin
(e.g. Sigma C3416): Prepare a 10 mg/ml stock (100x) in sterile
water and pass through a 0.2 µm filter.
Aliquot and store at -20°C.
Dilute into broth or agar-medium (cooled to 55°C) to a
final concentration of 100 mg/ml.
Luria Bertani medium (Lennox, unsupplemented)
per Invitrogen Recommendation for Selective Growth-
The standard LB medium contains 10 g
peptone, 5 g yeast extract and 10 g NaCl per liter.
- Weigh 10 g of LB broth base
(Invitrogen #12780-052) into a 1 liter Pyrex bottle
- Add d.i. water to 500 ml
- Microwave and mix until dissolved.
- Check pH and adjust to pH
7.5 as needed.
- Autoclave 15 psi, 121°C
for 15
minutes.
- Cool to room temperature.
- Aliquot into tubes/flasks
for growth and add antibiotics for selection
- Zeocin stock is 100 mg/ml;
dilute 1/4000 for 25 mg/ml
final
- e.g. 6.25 µl
per 25 ml medium in shake flask
- Carbenicillin stock is 10
mg/ml; dilute 1/100 for 100 µg/ml
final.
- e.g. 250 µl
per 25 ml medium
- In a 200 ml Pyrex bottle,
add 2 grams LB broth base (Invitrogen),
- Add d.i. water to 100 ml
- Check/adjust pH and add 1.5
g agar (Bacto 214010).
- Microwave and mix until agar
is melted and dissolved.
- Autoclave 15 psi, 121°C
for 15
minutes.
- Cool to 55°C
and add selective antibiotics-
- Zeocin (12.5 µl of
100 mg/ml stock per 100 ml agar/medium)
- Carbenicillin (0.5 ml of 10
mg/ml stock)
- Mix and pour ~20 into 100
x 15 mm Petri dishes (Fisherbrand 08-757-12).
(When fewer selective plates are
needed, transfer the required amount of agar/broth to a sterile 50 ml
conical tube before adding antibiotics. Use the remaining agar/broth
to pour standard LB plates. e.g. 6.25 µl
Zeocin and 250 µl Carbenicillin.)
Qiagen- Culture and Plasmid Purification
Though Qiagen recommends Luria Bertani
medium (e.g. LB Miller, with 10g peptone, 5g yeast extract and 10g NaCl
per liter) we use the Lennox low salt (5g/liter) broth base to improve
the stability of the Zeocin selective antibiotic. Single colonies
should be grown under selection followed by production of inoculum culture
and finally a production culture grown to full-log 3-4E9 cells per ml
or about 3 g wet weight per liter).
Grow bacteria-
- Pick a single colony from
a freshly streaked selective plate (e.g. with Zeocin & Carbenicillin,
25 mg/ml
each) and streak second selective plate.
- Pick a single colony from
the second plate, and inoculate 2 ml LB broth (with 25 µg ea Zeocin and carbenicillin); incubate at 35°C
300 rpm
for 8 hr.
- Transfer 50 µl
of the inoculum culture to 25 ml LBzc broth
in a 125 ml baffled shake flask, and incubate 25°C
300 rpm for 12-16 hr.
Prepare reagents-
- Add Lyse Blue dye (1000x stock)
to Buffer P1 (Tris-HCl, EDTA, pH 8) as needed, and add RNAse (100 mg/ml,
1000x stock for a final concentration of 100 µg/ml).
Prepare 4 ml for each plasmid (midi)
prep.
- Pre-chill neutralization buffer
P3 (potassium acetate, pH 5.5).
- Warm buffer P2 (1% SDS + 0.2M
NaOH) if precipitate is present.
Extract and purify DNA (Qiagen
Plasmid Midikit, Cat. No. 12143)-
- Transfer the bacterial cultures
to a 50 ml polypropylene tube (Nalgene Oak Ridge 3119-0050) and centrifuge
in Sorvall RC5 using the SA600 rotor at 8,000 rpm (6k x g) 15 min 4°C.
Discard supernatant. (Pellets may be stored at -20°C
pending
further processing.)
- Mixed the chilled Buffer P1
(Tris-HCl, EDTA, pH8 with RNase A) so that the LyseBlue is evenly suspended,
and transfer 4 ml to each pellet. Resuspend the pellet(s)
by vortexing or pipetting up and down until all clumps are dispersed.
- Add 4 ml of buffer P2 (SDS/NaOH),
and mix by vigorous inversion of the sealed tube 4-6 times; stand at
room temperature (15-25°C) for 5 minutes.
(Do not vortex
or leave more than 5 minutes as this will promote shearing of the genomic
DNA.) Cap buffer P2 immediately to avoid adsorption of CO2.
The solution should be a homogeneous blue color at this point.
- Add 4 ml of chilled buffer
P3 (potassium acetate, pH 5.5) and mix immediately by inverting 4-6
times; incubate on ice for 15 minutes. Note, the viscosity of
the solution should decrease as the genomic DNA, protein, cell debris
and potassium dodecyl sulfate (KDS) are precipitated. Every trace
of the LyseBlue should disappear.
- Centrifuge 13,000 rpm (20k
x g) 30 minutes 4°C
in the SA600 rotor. Remove the supernatant (containing the plasmid
DNA) promptly, and transfer to a fresh tube.
- Centrifuge 13,000 rpm for
15 minutes 4°C,
and remove the supernatant promptly. Save a 240 µl
aliquot for gel analysis.
- Equilibrate a Qiagen-tip 100
by applying 4 ml buffer QBT (0.75 M NaCl, MOPS, pH 7, 15% IPA) and allow
to empty by gravity flow.
- Apply the plasmid-containing
supernatant from the second spin to a Qiagen-tip and allow to enter
by gravity flow. Save 240 µl of
flow-through for gel analysis.
- Wash the Qiagen-tip with 2
x 10 ml buffer QC (1M NaCl, MOPS, pH 7, 15% IPA). Save 400 µl of the
combined wash fractions for gel analysis.
- Elute DNA using 5 ml buffer
QF (1.25 M NaCl, Tris-HCl, pH 8.5, 15% IPA). Collect the eluate
in a 15 or 50 ml polypropylene tube. Save 100 µl
eluate for gel analysis.
- Add 3.5 ml (i.e. 0.7 volumes)
room temperature isopropanol (IPA) to the eluted DNA. Mix and
centrifuge immediately at 13,000 rpm, 4°C.
Carefully remove the supernatant.
- Wash the pellet with 2 ml
room-temperature 70% ethanol, and centrifuge at 13,000 rpm at 4°C.
- Air-dry the pellets for 5-10
minutes, and redissolve the DNAin TE buffer, pH 8.0 or 10 mM Tris-Cl,
pH 8.5
DNA Quantification-
Note,
A260 for purified DNA should be 1.0 for a 50 µg/ml
solution
at neutral pH.
- Prepare a dilution of DNA
in Tris-Cl, pH 7 (aim for 5-50 µg/ml).
The Eppendorf UVette disposable
cuvette can be used with as little as 50 µl,
though 100 µl may be preferable.)
- µg
DNA/ml = OD260 x 50 x dilution
Agarose Gel Electrophoresis-
Gel preparation-
- Prepare TAE (tris-acetate
EDTA) buffer
- 50x: 242 g Tris base,
57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA, pH 8 (final volume of
1 liter brought up with distilled water).
- 1 x: 20 ml 50x up to
1 liter with d.w.
- Ethidiuim bromide stock, 10
mg/ml in d.w. (store at 4°C in
light-proof bottle).
- 6x Gel loading buffer:
In a 15 ml conical tube, add 25 mg bromphenol blue, 25 mg xylene cyanol
FF and 4 g sucrose (or 1.5 g Ficoll 400) and dissolve in d.w. to a final
volume of 10 ml. Store at 4°C (or RT for
ficoll-containing buffer).
- Prepare sufficient agarose
(e.g. Invitrogen UltraPure 15510-027)
- 50 ml required for small gel
box (8 x 10 cm appx). Use 0.8% agarose for 11.7 kb plasmid.
- Weigh 0.4 g agarose into a
200 ml beaker; suspend in 50 ml 1x TAE; mark level, and cover loosely
in plastic wrap. Microwave until agarose is melted/dissolved,
and the solution is just beginning to boil.
- Cool on the bench (or in a
water bath) to 55-60°C. Replenish any
volume that may have been
lost by addition of d.i. water, and add 2.5 µl
of 10 mg/ml ethidium bromide stock.
- Place a glass plate inside
the gel box and place the comb 1 cm from one end of the box, allowing
0.5-1 mm clearance from the base and pour the agarose solution into
the gel box to a depth of ~3-5 mm.
- When the agarose is solidified,
add a small amount of TAE buffer to the surface of the gel, and remove
the comb. Using a pipet tip, push up on the glass bottom, and
transfer the glass slide with agarose gel to the running box.
Add sufficient TAE to fill the electrode chambers and to cover the gel
with 1 mm depth buffer. Ethidium bromide may also be added to
the TAE running buffer (to 0.5 mg/ml).
- Prepare samples- 0.2-0.5 µg of plasmid
DNA can be loaded per lane. (As little as 20 ng may be detectable.)
- In an Eppendorf tube add DNA
and 1x TAE (10 µl
combined volume) followed by addition of 2 ul gel loading buffer.
- Rinse the sample wells with
1X TAE, and apply the samples
- Connect electrodes so that
the anode is on the opposite end of the gel box from the sample wells.
Turn on the power supply, applying 1-10 V/cm until the tracking dyes
indicate adequate separation. (Running voltage for the small gel
box would be 18-180 V. Starting with 50-100 volts should be adequate.)