Work Instruction
WI0017
Rev: A
Linearization of Supercoiled DNA for the Stable Transfection of Cell Lines
1. Materials
1.1. Plasmid with cDNA inserted
1.1.1. Guidelines: pick a plasmid to insert your cDNA into based on which
selectable marker and promoter you would like to use. This should already
be prepared before using this protocol.
1.1.2. Example: pcDNA3.1(+)
1.2. Restriction Enzyme
1.2.1. Guidelines: pick a restriction enzyme that will cleave your plasmid in one
location. Check the manual for the plasmid you are using for the
manufacturer’s recommendations and verify the enzyme will not cleave the
inserted DNA sequence.
1.2.2. Note: Restriction enzymes are generally supplied in glycerol and should
be stored at -20 ºC. Therefore, do not worry about freeze/thaw cycles
damaging the protein when placing in and out of the -20 ºC. Keep the
restriction enzyme stock cooled at all times.
1.2.3. Verify the recommended working temperature and concentration for your
restriction enzyme. Examples:
1.2.3.1.Ssp I
1.2.3.1.1. Manufacturer: New England Biolabs
1.2.3.1.2. Concentration: 0.2 U/µL
1.2.3.1.3. Working Temperature: 37 ºC
1.2.3.2.Sph I
1.2.3.2.1. Manufacturer: New England Biolabs
1.2.3.2.2. Concentration: 0.2 U/µL
1.2.3.2.3. Working Temperature: 37 ºC
1.3. Restriction Enzyme Buffer
1.3.1. Guidelines: be sure to pick a buffer that has 100% activity with your
enzyme. Most likely the enzyme will come with such a buffer.
1.4. TE Buffer
1.4.1. Composition: 10 mM Tris & 1 mM EDTA
1.4.2. Note: Qiagen elution buffer (EB) is essentially pre-made TE buffer and
can be used instead.
1.5. TAE Buffer
1.5.1. Composition: 40 mM Tris, 20 mM acetic acid & 1 mM EDTA. Adjust to
pH 8.0. For 500 mL of 50× stock, mix:
1.5.1.1.Tris, 121 g
1.5.1.2.Glacial acetic acid, 28.6 mL
1.5.1.3.EDTA, 50 mL of 0.5 M stock
1.5.1.4.Adjust to 500 mL total volume with ultrapure water and adjust pH to
8.0.
1.6. Sterile, RNAse, DNAse free eppendorf microcentrifuge tubes
1.6.1. Microcentrifuge tubes used for the protocol should come pre-sterilized and
certified free of RNAses and DNAses. Be sure to store in an autoclaved
container and keep the lid on when not in use.
1.7. Clean water
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Date: 2/08/08
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Work Instruction
WI0017
Rev: A
Linearization of Supercoiled DNA for the Stable Transfection of Cell Lines
1.7.1. RNAse, DNAse free water is preferable for the preparation of the
restriction solution, although Milli-Q filtered water can be substituted if
none is available.
1.8. Mini Submersion Cell Electrophoresis Machine
1.8.1. Manufacturer: BioRad
2. Procedure
2.1. Linearize the DNA.
2.1.1. Plan for 10 µg of DNA per 5 X 106 cells if establishing a cell line via
electroporation. You may wish to optimize this estimate for your cell line
and transfection process.
mDNA,desired,µg = 10 X Ntransfections (µg)
2.1.2. Plan on losing 10-40% of the DNA in the linearization process. Calculate
one volume equivalent for the protocol for a 0.5 µg/µL DNA suspension:
V = mDNA,desired,µg X 1.4 / 0.5 (µL)
2.1.3. Combine the following in the microcentrifuge tube:
2.1.3.1.Restriction buffer (10X), verify buffer will have 100% activity with
enzyme and does not require adding anything additional, such as BSA:
Vbuffer = 0.1 X V (µL)
2.1.3.2.DNA for digest:
Vundigested DNA = mDNA,desired,µg X 1.4 / Cundigested DNA, µg/µL (µL)
2.1.3.3.Calculate amount of restriction enzyme to add, but do not add the
enzyme yet. Usually 1 : 3 enzyme : buffer, depends on the enzyme
activity. For 100% activity, a working concentration of 0.2 Units / µL
frequently works well.
Vrestriction = 0.2 X V / (Crestriction stock, Units/µL)
2.1.3.4.Add sterile ddH2O. Use RNAse, DNAse free water if possible:
Vsterile H2O = V – Vundigested DNA - Vrestriction - Vbuffer
2.1.3.5.Add the restriction enzyme.
2.1.3.6.Mix gently. Do NOT vortex, just “flick,” DNA is especially sensitive
to shearing prior to when the linearization step is complete.
2.1.3.7.Centrifuge using a microcentrifuge at low speed for a couple of
seconds, just a pulse to get everything to the bottom of the tube.
2.1.4. Incubate at 37 ºC for at least 2 hr, it is OK to go longer. Verify this is
appropriate for your enzyme.
2.2. Run an agarose gel to verify the restriction was successful.
2.2.1. Prepare the gel
2.2.1.1.Agarose (1%) in TAE buffer (0.6 g agarose + 60 mL TAE). Add 10
µL of ethidium bromide (10 mg/mL stock).
2.2.1.2.Microwave ~1 min, turns clear when ready.
2.2.1.3.Pour onto the gelcaster and allow to solidify. The gel should cool in
about 30 minutes.
2.2.2. Load the gel
2.2.2.1.Place the gel in the electrophoresis machine and pour TAE buffer over
the gel so it is submerged.
2.2.2.2.Prepare the samples:
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Date: 2/08/08
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Work Instruction
WI0017
Rev: A
Linearization of Supercoiled DNA for the Stable Transfection of Cell Lines
2.2.2.2.1. Molecular weight ladder for lane 1, 1 µg of ladder DNA. This
is 5 µL of ladder in gel loading buffer at 0.2 µg/mL.
2.2.2.2.2. At least 0.5 µg digested DNA sample (1 µL) and enough TE
buffer for 10 µL total volume. Add 2 µL of gel loading buffer.
2.2.2.2.3. An equivalent amount of plasmid DNA and enough TE buffer
for 10 µL total volume. Add 2 µL of gel loading buffer.
2.2.3. Load the samples and run the gel: 60V for about 45 min. Longer if
necessary, inspect the loading buffer migration. The front need only travel
about 3-4 cm to achieve sufficient separation for analysis.
2.2.4. Image the gel using UV transillumination for ethidium bromide. Note the
linearized plasmid should not migrate as far as the supercoiled if the cut was
successful. The linearized DNA should migrate as a single band. Sample:
MW Marker Linearized Supercoiled
2.3. Clean up the DNA.
2.3.1. If the volume is less than 500 µL, dilute to 500 µL with TE buffer.
2.3.2. Phenol extraction
2.3.2.1.Add an equal volume of tris-saturated phenol (phenol on bottom layer)
solution to the DNA sample.
2.3.2.2.Vortex briefly, the solution should turn a cloudy white.
2.3.2.3.Spin down the sample, 10,000 RPM for 2 min at 4 ºC. Not sensitive to
precise centrifugation speed.
2.3.2.4.Recover top aqueous phase (possibly cloudy), but don’t recover any
phenol, better to lose some sample than trying to recover entire aqueous
layer. A concentrated protein layer may be visible at the water/phenol
interface.
2.3.3. Chloroform extraction to remove phenol
2.3.3.1.Add 1 volume of 24:1 choloroform : isoamyl alcohol to 1 volume of
recovered aqueous phase to extract remaining phenol from sample.
The 24:1 mixture may be be refrigerated.
2.3.3.2.Vortex briefly.
2.3.3.3.Spin down the sample, 10,000 RPM for 2 min at 4 ºC. This step is not
sensitive to precise centrifugation speed.
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Date: 2/08/08
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Work Instruction
WI0017
Rev: A
Linearization of Supercoiled DNA for the Stable Transfection of Cell Lines
2.3.3.4.Recover the top layer, don’t recover any of the bottom layer. TE
buffer can be added to the remaining sample and the mixture can be
centrifuged again to improve quanitative recovery.
2.3.4. ETOH precipitation:
2.3.4.1.FIRST add 3M sodium acetate, pH 5.4, as 0.1 X V to the recovered
DNA sample (Adjust the V value if necessary for < 100% recovery).
2.3.4.2.Add 100% EtOH, as 2.5 X V.
2.3.5. Give this a quick vortex.
2.3.6. Incubate at decreased temperature to allow precipipation:
2.3.6.1.Liquid Nitrogen: would take approximately 2 minutes.
2.3.6.2.–80 oC: would take approximately 15 minutes.
2.3.6.3.–20 oC: would take approximately 1 hr.
2.3.7. Spin the EtOH/DNA sample at 16,000 x g for 10 min at 4 ºC.
2.3.8. The DNA will be a white pellet at the bottom. Pour off as much ethanol
as possible and add 70% ethanol in milli-Q water to wash the pellet. The
pellet will not go into solution, this step removes the salt.
2.3.9. Shake or vortex.
2.3.10. Spin 16,000 x g for 2 min.
2.3.11. Pour off the supernatant and make sure the pellet stays at the bottom.
2.3.12. Let the container sit open for a while in a sterile environment so the pellet
can dry. It is OK to fully dry the pellet, this requires more time than partial
drying, but requires less judgement and does not appreciably harm the DNA.
2.3.13. Once the ethanol is gone add TE buffer so the DNA is at roughly 1 µg/mL
based on the starting amount:
Vfinal = 0.5 X V
2.4. Store at 4 ºC, or at -20 ºC if it will be > 1 month before use. Be sure to determine
the final DNA concentration and purity based on the A260 and A280 with a
spectrophotometer.
3. References
3.1. Restriction Enzymes
3.1.1. New England Biolabs Website: www.neb.com
3.2. Plasmids
3.2.1. Invitrogen Website: www.invitrogen.com
3.3. Personal communications and training
3.3.1. Konkal-Matt R. Prasad, Ph.D., University of Virginia
3.3.2. Karen R. Snapp, Ph.D., University of Illinois
4. Revision History
Date
Rev Description
2/08/08 Initial release.
10/10/08 A
Updated detail on buffer and gel
preparation.
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Date: 2/08/08
Revision Author
Brian J. Schmidt
Brian J. Schmidt
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