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Cleavage of Vector DNA with Restriction Enzyme(s) and Removal of "Stuffer" DNA
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol illustrates processing of vector DNA using fUSE5 as an example. The fUSE5 vector has two SfiI sites separated by a 14 base pair segment of "stuffer" DNA. The stuffer is removed by filtration through an Amicon filter with a 100 KDa molecular weight cutoff (Centricon 100). The protocol does not use calf instestine phosphatase to remove 5' phosphates from the cleaved vector because dephosphorylation may be inefficient with SfiI produced 3' overhangs. Furthermore, the two SfiI ends are neither complementary nor self-complementary and therefore cannot be ligated to themselves or each other.
Procedure
A. Vector Cleavage
1. Add in the following order to a 1.5 ml microcentrifuge tube:
X μl ddH2O (add first)
360 μg fUSE5 Replicative Factor
144 μl of 10X React 2 Buffer
1440 Units of Restriction Enzyme SfiI
Add an appropriate volume of ddH2O to give a final volume of 1.44 ml.
2. Vortex the tube gently, microcentrifuge briefly to drive the solution to the bottom of the tube, and then incubate at 50° for 2 hr.
3. Add 72 μl of 250 mM EDTA to stop the digestion.
4. Aliquot the digestion solution into three equal parts by transferring 504 μl (one-third the solution) into a fresh 1.5 ml microcentrifuge tube.
5. Add an equivalent volume of Neutralized Phenol to each tube and mix vigorously.
6. Extract with the double-centrifugation method (see Hint #1):
a) microcentrifuge briefly at maximum speed to separate the phases
b) using a 200 μl pipette, carefully draw off the organic (lower) phase of the solution.
Leave all of the interphase and upper phase in the microcentrifuge tube (see Hint #2).
Microcentrifuge at maximum speed to re-separate the phases.
Transfer the upper (aqueous) phase to a fresh 1.5 ml microcentrifuge tube, taking care to avoid any contamination with the interphase or lower (organic) phase.
7. Add an equal volume of 100% Chloroform to the aqueous phase, mix vigorously and collect the aqueous phase using the double-centrifugation method (Step #A6).
8. Degas the three tubes for approximately 30 min in a vacuum centrifuge (such as a SpeedVac) to get rid of any trace of chloroform (see Hint #3).
9. Pool all three aqueous phases into a single graduated tube and add TE Buffer to bring the total volume to 4 ml.
10. Add one half of the solution to each of two Centricon 100s.
11. Centrifuge the Centricon filters at 2,500 rpm using a Sorvall™ SS-34 rotor (700 X g) at 4° until the volume remaining in the Centricon 100 well is approximately 100 μl (approximately 1 hr).
12. Discard the pass through volume and add sufficient TE Buffer to bring the volume in the well of the Centricon 100 filter to 2 ml.
13. Centrifuge the Centricon filters at 2,500 rpm in a Sorvall™ SS-34 rotor (700 X g) at 4° until the volume remaining in the Centricon 100 well is approximately 100 μl.
14. Repeat the TE Buffer washing steps (Steps #12 and #13) five more times (see Hint #4).
15. After the final concentration step, add 150 μl TE Buffer to each of the two retentates, cover the Centricon with parafilm, and vortex to ensure mixing of the concentrated DNA solution.
16. Collect the diluted retentates by back-centrifugation into a collection tube according the manufacturer's (Amicon) instructions.
17. Pool the back-centrifugation solutions into a single tarred 2 ml microcentrifuge tube.
18. Add an additional 150 μl of TE Buffer to each of the filters, vortex, and collect these washes by back-centrifugation.
19. Pool all the washes into the single tarred 12 ml microcentrifuge tube and estimate the weight of the solution (weight in g = volume in ml).
20. Add additional TE Buffer as necessary to bring the total volume to 1.5 ml and store at 4°C (see Hint #5).
21. Prepare 200 μl of a 1:30 dilution of the original sample.
22. Scan the absorbance from 220 to 300 nanometers (see Protocol ID#2174 and Hint #6).
B. Electrophoresis of the Digested Sample
1. Mix 20 μl of the 1:30 dilution (approximately 160 ng of DNA) in a 500 μl microcentrifuge tube containing 3.3 μl of 70/75/BPB Solution.
2. Electrophorese the sample on a 8% Agarose/ 4X GBB gel along with suitable DNA markers (such as λ.HindIII or λ.BstEIII markers, also see Hint #7).
C. Verification of Ligation
1. Combine 300 ng of the fUSE5 digest (Step #A20) with 3.75 μl of 10X Commercial Ligation Buffer (see Hint #8). Add ddH2O to bring the final volume to 37.5 μl.
2. Mix and evenly divide the solution into three 1.5 ml microcentrifuge tubes (12.5 μl is one-third volume).
3. To Tube #1, add 1 ng of 20 to 50 base pair synthetic test insert (see Image #1) in 1 μl of TE Buffer or other non-interfering buffer.
4. To Tubes #2 and #3 add 1 μl of TE Buffer.
5. On ice, dilute T4 DNA Ligase in sufficient Ligation Buffer to bring the final enzyme concentration of approximately 10 Weiss Units/ml.
6. Add 11.5 μl of the T4 DNA Ligase solution to Tube #1 and Tube #2. Add 11.5 μl of 1X Ligation Buffer to Tube #3.
7. Incubate for 6 to 16 hr in a 10° water bath (see Hint #9).
D. Electrophoresis of Ligated Sample
1. Add 4.2 μl 70/75/BPB to each of the three samples and mix well (see Hint #10).
2. Electrophorese the sample on a 8% Agarose / 4X GBB gel next to a suitable DNA size marker (such as λ.HinDIII or λ.BstEIII markers, also see Hint #11).
Solutions
GBB (40X)
Store at room temperature
Dissolve in approximately 500 ml ddH2O
18.83 g Disodium EDTA
45.94 g Anhydrous Sodium Acetate (or 76.16 g Trihydrate)
Adjust the pH to 8.3 with Glacial Acetic Acid
Adjust the final volume to 700 ml with ddH2O
142.4 g Tris-HCl 
70/75/BPB Solution
70% (v/v) Glycerol
75 mM EDTA
0.3% (w/v) Bromophenol Blue
TE Buffer
Autoclave and store at room temperature
10 mM Tris-HCl, pH 8.0
1 mM EDTA
1 M Tris-HCl, pH 8.0
Neutralized Phenol
Allow phases to separate and remove the aqueous (upper) phase
Equilibrate with Tris once more
Use water-saturated Phenol (CAUTION! See Hint #19)
Shake or vortex vigorously to equilibrate phases
Add one-tenth volume of 1 M Tris-HCl, pH 8.0
Use the lower phase as Neutralized Phenol
250 mM EDTA
BioReagents and Chemicals
Sodium Acetate
Ethidium Bromide
Bromophenol Blue
Acetic Acid
Glycerol
Chloroform
Phenol
Restriction Enzyme, SfiI
React 2 Buffer
Oligonucleotide
DNA Electrophoreses Markers, λ.HindIII
EDTA
Tris-HCl
DNA Ligase, T4
DNA Electrophoreses Markers, λ.BstEIII
Protocol Hints
1. The contributor advocates using the double-centrifugation method, which greatly increases the yield of the aqueous phase from each organic extraction.
2. The purpose of removing the organic phase is to lower the interphase into the narrow tip of the microcentrifuge tube so that the aqueous phase can be drawn off with a high yield. Avoid removing the aqueous phase.
3. The Centricon 100 to be used in the next step is not resistant to residual chloroform. The contributor of the protocol notes that although a residual odor of chloroform may remain after the degassing, the Centricon 100 seems to withstand this small amount.
4. This entire washing procedure may take between 1 to 2 days to complete.
5. The nominal DNA concentration, assuming 100% yield, is approximately 240 ęg/ml.
6. The nominal A268 (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.
7. Linearized fUSE5 (the intented product) is 9.2 Kbp long and essentially co-electrophoreses with the 9.4 Kbp λ.HindIII band (just behind the 8.45 Kbp λ.BstEII band); uncut fUSE5 Replicative Factor electrophoreses as if it were a linear fragment of approximately 5 Kbp, while open circular fUSE5 Replicative Factor electrophoreses as if it were a linear fragment of approximately 11 Kbp. Only the linear band should be visible. The gel 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 base pair stuffer DNA has been effectively removed. This is why the ligation test in Section C is performed. This test requires a synthetic test insert (see Image #1).
8. Use any appropriate commercially available 10X Ligation Buffer that contains ATP. Do not use a Ligation Buffer that contains Polyethylene Glycol. Each tube should contain 100 ng of vector DNA in 12.5 μl of 1X Ligation Buffer.
9. Fill a well-insulated ice bucket fairly full with 10°C water, add the microcentrifuge tubes to incubate and leave the ice-bucket covered at 4°C.
10. The test insert has phosphorylated 5' ends, so that the ligation creates covalently closed circular (but not supercoiled) ligation products. These products consist of approximately 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, either use test inserts with 5' hydroxyls or include 0.5 μg/ml Ethidium Bromide in the gel. The Ethidium Bromide induces positive supercoiling in the covalently closed circular ligation products, making all seven or so topoisomers migrate with similar mobility, as if they were approximately 5 Kbp linear double-stranded fragments. In this example, the test insert is not phosphorylated, so that the intended ligation product is open circular DNA.
11. Add 0.5 μg/ml Ethidium Bromide if necessary; see Hint #10. Linear vector (without insert, or with an insert joined only at one end) electrophoreses as a 9.2 Kbp linear double-stranded molecule. Open circular vector (presumably containing an insert) electrophoreses as an approximately 11 Kbp linear double-stranded molecule. Sometimes slower bands corresponding presumably to higher multimers are visible. The vector is considered suitable if only linear vector appears in the ligation without insert (Tube #2) and in the mock ligation without ligase (Tube #3); whereas a substantial amount of open circular vector (at least 10%) appears in the ligation with test insert (Tube #1).
