To study the desired functionality of an insert there is a requirement of transferring them from one vector to another, this procedure of transferring is known as subcloning. Steps of the Subcloning:
- Release and purify your insert from the parent vector
- Ligate this insert into a prepared destination vector
- Transform this ligation reaction into competent bacterial cells
- Screen the transformed cells for the insert
Different Subcloning Strategies
For any work on subcloning some prerequisite knowledge about the vector and insert is necessary. The information about the RE sites available in the parent vector multiple cloning regions, RE sites available the insert (in case of inset digestion). Also information about the recurrence of RE sites in destination vector multiple cloning regions and RE sites in the insert. Depending on the different combination of these sites, the subcloning strategy to be used is decided.
Subcloning Strategy: Common Restriction Sites
A straightforward subcloning process can be utilized if the parent and destination vector multiple cloning regions contain common restriction sites and neither of these restriction sites occurs within your insert. Both the parent and destination vectors are digested using same two enzymes. This is followed by dephosphorylation of the destination vector. Separate both the insert and the dephosphorylated vector using an agarose gel. Then purify insert vector using any DNA purification system. Finally, these vectors and inserts are ligated.
Subcloning Strategy: Moving Inserts with Compatible Restriction Sites
There are cases when restriction sites in the parent and destination vector multiple cloning regions are not common but may be compatible. Compatible restriction sites have the same overhang sequence and can be ligated together. After ligation, the regenerated sites does not resemble both restriction sites in the parent and destination vector multiple cloning regions. Once the necessary enzymes for the ligation and restriction are identified. This cloning strategy also becomes straightforward.
Subcloning Strategy: Moving Inserts with Only One Common Site
The combination of common restriction sites leads to a possibility to only one match on one side of your insert. The issue should be addressed on the other side of the insert. A cut-blunt-cut can be used. The action of T4 DNA Polymerase can blunt any restriction site. Digest the parent vector and blunt that site with T4 DNA Polymerase. Run the products on a gel, purify and proceed with the common or compatible end restriction enzyme digestion. Most vectors have at least one blunt-ended restriction site that can accept the newly created blunt end from the insert. If you don’t have such a site or the site would not be in the correct orientation, the same “cut-blunt-cut” strategy may be applied to the destination.
Subcloning Strategy: Blunt-End Method
The final scenario of no common restriction site is common or compatible with the parent or destination vector. IN this scenario the best strategy is to amplify the insert with restriction sites in the primers to provide the compatibility. But this method has an own set of problems. There is a possibility of mutations. Also, there is a lot of difficulty in the digestion of the PCR products. One more strategy can be used in this scenario. Cut out the insert with any enzymes. Treat with T4 DNA Polymerase to blunt either 5′ or 3′ overhangs and ligate into the destination vector opened with a blunt-end cutter or made blunt by T4 DNA Polymerase. Orientation of the isert may not be retained by this method.
Methods
Restriction Digestion
Restriction endonucleases (RE), also referred to as restriction enzymes, are proteins that recognize short, specific (often palindromic) DNA sequences. Type II REs cleave double-stranded DNA (dsDNA) at specific sites within or adjacent to their recognition sequences. Many restriction enzymes will not cut DNA that is methylated on one or both strands of the recognition site, although some require substrate methylation. Restriction digestion is one of the most common reactions performed in molecular biology.
- For a digestion with a single RE the reaction is very simple:
Nuclease-Free Water 14μl
10X Restriction Buffer 2μl
Acetylated BSA (1mg/ml) 2μl
DNA (~1μg) 1μl
Restriction Enzyme (10u) 1μl
Final Volume 20μl
Mix by pipetting and collect the contents at the bottom of the tube. Incubate at the appropriate temperature for the enzyme for 1–4 hours. Add 4μl of 6X Blue/Orange Loading Dye and analyze digested DNA by gel electrophoresis.
- Preparing an insert for transfer from one vector to another usually requires a double digest (digest with two different REs). If both restriction enzymes work in the same restriction enzyme buffer, the reaction is straightforward. Simply add 1μl of the second restriction enzyme and adjust the amount of water used. Remember, restriction enzymes are commonly stabilized in 50% glycerol solution. Do not exceed 5% glycerol in final digest with the two enzymes. Glycerol concentrations >5% may lead to star activity.
Partial Restriction Digestion
Controlling Cut Frequency in Restriction Digestion
The presence of a restriction recognition site in the insert and the multiple cloning region does not necessarily preclude use of that restriction site in a subcloning strategy. Under normal restriction digest conditions, the enzyme is in excess so that all recognition sites in the plasmid can be cleaved. You can manipulate the restriction digest conditions such that you will digest only a subset of sites. Many strategies have been employed to do partial digests: Decreasing reaction temperature, using a non-optimal buffer, and decreasing units of enzyme. The method presented here uses dilutions of enzyme in the optimal buffer
- Digest 10μg of parent vector to completion to linearize (i.e., RE1; 50μl reaction).
- Purify vector with the Gel and PCR Clean-Up System directly from the reaction. Elute in 20μl nuclease-free water.
- On ice, create serial dilutions of RE2 in 1X RE Buffer containing 0.1mg/ml Acetylated BSA (e.g. to yield 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039u of RE per 18μl of solution).
- Add 2μl of the purified vector to each tube.
- Incubate all reactions at 37°C for 30–45 minutes.
- Add loading dye to each reaction and analyze digests by agarose gel electrophoresis.
- Identify and cut bands from the gel containing the DNA fragment of interest.
- Purify insert using the Gel and PCR Clean- Up System. Elute in 15–20μl nuclease-free water.
- Proceed to ligation reaction.
Dephosphorylating Vectors to Limit Self-Ligation
Preventing vector self-ligation is critical for reducing subcloning background. The efficiency of ligating the plasmid to itself is far better than ligating a separate piece of DNA into the vector and is the favored reaction. Removing the 5′ phosphates of the linearized vector will prevent T4 DNA Ligase from recircularizing the vector. Calf Intestinal Alkaline Phosphatase is the classic enzyme for vector dephosphorylation. The enzyme can be used on 5′ recessed ends (i.e., results from an enzyme leaving a 3′ overhang), 5′ overhangs and bluntends. After dephosphorylation, the enzyme must be removed either by direct purification or gel electrophoresis and gel isolation with DNA purification systems like the Gel and PCR Clean-Up System. Shrimp Alkaline Phosphatase can be used in place of Calf Intestinal Alkaline Phosphatase and offers the advantage of simple heat denaturation to inactivate the enzyme without the need for further purification.
Dephosphorylating Vectors: Shrimp Alkaline Phosphatase Streamlined Restriction Digestion, Dephosphorylation and Ligation Procedure
- Combine restriction digestion and dephosphorylation of DNA vector in 1X restriction enzyme buffer. Use 15 units of restriction enzyme/μg vector and 10 units Shrimp Alkaline Phosphatase (SAP)/μg vector in a final volume of 30–50μl. Incubate at 37°C for 15 minutes. This is a sufficient amount of SAP to completely dephosphorylate the vector regardless of overhang type (5′, 3′, or blunt) in any RE buffer.
- Heat-inactivate both restriction enzyme and SAP for 15 minutes at 65°C.
- Centrifuge and remove 1–2μl of vector for ligation with appropriate DNA insert using T4 DNA Ligase and 2X Rapid Ligation Buffer from Rapid DNA Ligation System at 15°C for 5 minutes (3′ or 5′ ends) or 15 minutes for blunt ends in a final reaction volume of 10–50μl. We recommend starting with a 1:2 molar ratio of vector:insert DNA.
- Transform the ligated material directly into competent E. coli cells.
Purifying Vector and Insert
Purification of the insert and destination vector are absolutely critical for success in subcloning applications. Years ago, each step called for phenol:chloroform extractions followed by ethanol precipitation to remove enzymes such as calf intestinal alkaline phosphatase from enzymatic vector manipulations. Guanidine-based nucleic acid clean-up systems greatly simplified the removal of enzymes. Gel isolation methods further improved the efficiency of subcloning by segregating the wanted reactants from the unwanted reactants.
Purifying Vector and Insert
Gel and PCR Clean-Up System
Gel and PCR Clean-Up System is designed to extract and purify DNA fragments directly from PCR(a) or from agarose gels. Fragments of 100bp to 10kb can be recovered from standard or low-melt agarose gels in either Tris acetate (TAE) buffer or Tris borate buffer (TBE). Up to 95% recovery is achieved, depending upon the DNA fragment size. This membrane-based system, which can bind up to 40μg of DNA, allows recovery of isolated DNA fragments or PCR products in as little as 15 minutes, depending on the number of samples processed and the protocol used. Samples can be eluted in as little as 15μl of nucleasefree water. The purified DNA can be used for automated fluorescent sequencing, cloning, labeling, restriction enzyme digestion or in vitro transcription/ translation without further manipulation.
Gel Electrophoresis
Agarose Gel Electrophoresis of DNA
Running double-stranded, linear DNA (like plasmid DNA from restriction enzyme digests) on an agarose gel is a routine activity in molecular biology laboratories. The basic method is very straightforward:
- Set up the minigel apparatus as recommended by the manufacturer.
- Weigh the required amount of agarose and add it to the appropriate amount of TAE or TBE 1X Buffer in a flask or bottle. For example, to prepare a 1% agarose gel, add 1.0g of agarose to 100ml of buffer. The volume of buffer and agarose should not exceed half the volume of the container.
- Heat the mixture in a microwave oven or on a hot plate for the minimum time required to allow all the agarose to dissolve. Interrupt the heating at regular intervals and swirl the container to mix the contents. Do not allow the solution to boil over.
- Cool the solution to 50–60°C and pour the gel. Allow the gel to form completely (typically, 30 minutes at room temperature is sufficient). Remove the comb from the gel, place it in the electrophoresis chamber and add a sufficient volume of TAE or TBE 1X buffer to just cover the surface of the gel.
- Load samples with 1X Blue/Orange Loading Dye into the wells.
- Connect the gel apparatus to an electrical power supply and apply an appropriate voltage to the gel. For minigels, typical gradients used are between
1–5 volts/cm. Higher voltages and shorter runs will decrease the resolution of the gel and may also cause overheating that may melt the agarose.
- After electrophoresis is complete, remove the gel and stain it by soaking it in a solution of 0.5μg/ml ethidium bromide for 30 minutes at room temperature. Note: Ethidium bromide may also be incorporated in the gel and electrophoresis buffer, at a concentration of 0.5μg/ml, during gel preparation. This eliminates the need for post-electrophoretic staining but may interfere with accurate size determination of DNA fragments.
- Place the gel on a UV lightbox and photograph the gel according to the specification recommended for your camera and film type. CAUTION: Use protective eyewear when working with a UV light source.
Materials
- Blue/Orange Loading Dye, 6X
10mM Tris-HCl, pH 7.5
50mM EDTA
15% Ficoll® 400
0.03% bromophenol blue
0.03% xylene cyanol FF
0.4% orange G
One or more dyes can be left out of the recipe to create a custom loading dye.
- TAE 50X Buffer (1L)
Dissolve 242g Tris base and 37.2g disodium EDTA, dihydrate in 900ml of deionized water. Add 57.1ml glacial acetic acid and adjust the final volume with water to 1 liter. Store at room temperature or 4°C.
- TBE 10X Buffer (1L)
Dissolve 108g of Tris base and 55g boric acid in 900ml deionized water. Add 40ml 0.5M EDTA (pH 8.0) and increase the final volume to 1L. Store at room temperature or 4°C.
DNA Markers
DNA markers should always be run on agarose gels to aid in identifying bands of interest. This is especially true if you are performing applications such as partial restriction digestion. Promega offers a wide variety of DNA markers to fit your needs. BenchTop Markers come premixed with Blue/Orange Loading Dye ready to load onto the gel. As the name implies, you can store them on your benchtop, no need to freeze and thaw every time you need it. Conventional markers are pure DNA solutions and come with a tube of 6X Blue/Orange Loading Dye for use with the marker and your samples.
Ligation: Ligating Vector and Insert
Molecular biologists have exploited DNA ligases to insert pieces of DNA into vectors for decades. The enzyme most commonly used is derived from bacteriophage T4. T4 DNA Ligase is about 400-fold more active than E. coli DNA ligase for ligating blunt ends, and thus is the enzyme of choice for all molecular biology requirements. Promega offers T4 DNA Ligase in standard or high-concentrate form, with the standard Ligase Buffer or with the 2X Rapid Ligation Buffer offered in the Rapid DNA Ligation System. The System allows rapid, 5-minute ligations for 5′ or 3′ overhang cohesive ends or 15-minute ligations for blunt ends.
Ligation
Rapid DNA Ligation System
Starting with a 1:2 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector is recommended.
The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses the 1:2 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA.
- Assemble the following reaction in a sterile microcentrifuge tube:
vector DNA 100ng
insert DNA 33ng
2X Rapid Ligation Buffer 5μl
T4 DNA Ligase (3u/μl) 1μl
nuclease-free water to 10μl
- Incubate the reaction at room temperature for 5 minutes for cohesive-ended ligations, or 15 minutes for blunt-ended ligations.
Ligation
T4 DNA Ligase
Using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector is recommended.
The following ligation reaction of a 3.0kb vector and a 0.5kb insert DNA uses the 1:3 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA.
- Assemble the following reaction in a sterile microcentrifuge tube:
vector DNA 100ng
insert DNA 50ng
Ligase 10X Buffer 1μl
T4 DNA Ligase (3u/μl) 1μl
Nuclease-Free Water to 10μl
- Incubate the reaction:
22–25°C 3 hours Cohesive ends
4°C Overnight Cohesive ends
15°C 4–18 hours Blunt ends
Ligation: Control Reaction
Controls help ensure that everything is functioning normally in your subcloning reaction. If something does go wrong, you can use your controls to figure out where a problem might have occurred. When ligating insert and vector, you can do a control ligation of vector with no insert. Carry this reaction through transformation and plating. The number of colonies you see can be a good indicator of how a ligation reaction performed and how many background colonies you will have on your plate.