Ligase independent cloning (LIC) is a simple, fast and relatively cheap method to produce expression constructs. It makes use of the 3'--> 5'-activity of T4 DNA polymerase to create very specific 10-15 base single overhangs in the expression vector. PCR products with complementary overhangs are created by building appropriate extensions into the primers and treating them with T4 DNA polymerase as well. The annealing of the insert and the vector is performed in the absence of ligase by simple mixing of the DNA fragments. This process is very efficient because only the desired products can form.
1. Preparation of vector DNA
The EMBL-made LIC vectors (see appendix for vectors maps) all contain the gene encoding for eGFP flanked by two BsaI sites (shown in red). These sites are used to linearize the vector, while at the same time removing the eGFP gene.
ATTTTCAGGGCGCCATGAGACCG..eGFP..GGTCTCACCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGTACTCTGGC..eGFP..CCAGAGTGGCGCAGCCCAGTGGTG
| BsaI
V
ATTTTCAGGGC...........................CCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGT...........................CAGCCCAGTGGTG
Next the digested vector is treated with T4 DNA polymerase in the presence of dTTP. Because of the 3'--> 5' activity of the polymerase the bases are removed from both 3'-ends until the first thymidine (T) residue is reached.
ATTTTCAGGGC...........................CCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGT...........................CAGCCCAGTGGTG
| T4 DNA polymerase + dTTP
V
ATTTT.................................CCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGT...................................TGGTG
This 2-step protocol leads to two specific overhangs in the LIC vector of 10 and 12 bases, respectively, which are allow the specific, ligase-independent annealing reaction (protocol 3).
1.1 Linearization of the LIC vector by BsaI digestion
Materials Chemicals
1.5-ml microfuge tubes agarose (electrophoresis grade)
6X Loading dye solution
MinElute Gel Extraction Kit ethidium bromide (10 mg/ml)
TBE buffer
Enzymes
BsaI
10X New England Biolabs buffer 3 (supplied with the enzyme)
Mix in a 1.5-ml microfuge tube:
5 µl 10X New England Biolabs buffer 3
5 µg LIC vector DNA
2.5 µl BsaI (10 units/µl)
Add sterile water to a volume of 50 µl
1. Add the restriction enzyme last
2. Mix by briefly vortexing the solution and spin 1 min at 13,000 rpm in a microfuge centrifuge.
3. Incubate the digestion mix for 1 hours at 50°C.
4. In the meantime, prepare a 0.8% agarose gel.
Dissolve 0.4 g agarose in 50 ml TBE buffer by heating. After the solution has cooled down add 1-2 µl ethidium bromide solution and pour it into a prepared gel running chamber. After the gel has solidified fill the chamber with TBE buffer.
5. Add 10 µl 6X loading dye solution to the sample. Mix well and spin 1 min at 13,000 rpm in a microfuge centrifuge.
6. Load the sample on the agarose gel.
7. Run the gel for 1 hours at 100 V.
8. Analyze the gel on a UV lamp and cut out the band of the linearized LIC vector.
Expose the gel as briefly as possible to the UV lamp to avoid damage to the DNA.
9. Purify the vector DNA form the gel pieces using the MinElute Gel Extraction Kit.
10. Elute the digested vector DNA in 50 µl elution buffer in a 1.5-ml microfuge tube.
The BsaI digestion does not necessarily work 100%. It is important to cut out the band of the linearized LIC vector carefully to minimize the amount of undigested vector in the final preparation, as this will give false positive results later on.
The concentration of vector DNA can be determined using the absorbance at 280 nm (assuming A280 = 1 is 50 ng/µl).
1.2 T4 DNA polymerase treatment of the linearized LIC vector
In the annealing protocol 25-50 ng prepared LIC vector is used per reaction (see protocol 3). In the following protocol 600 ng BsaI-digested LIC vector is treated with T4 DNA polymerase to produce enough vector for approx. 20 annealing reactions. This can be scaled up or down according to your own need.
Materials Chemicals
1.5-ml microfuge tubes dTTP (100 mM)
DTT (100 mM)
100X BSA
Enzymes
T4 DNA polymerase
10X New England Biolabs buffer 3 (supplied with the enzyme)
Mix in a 1.5-ml microfuge tube:
2 µl 10X New England Biolabs buffer 3
600 ng BsaI-digested LIC vector
0.5 µl dTTP (100 mM)
1 µl DTT (100 mM)
0.2 µl 100X BSA
0.4 µl T4 DNA polymerase (3 units/µl)
Add sterile water to a volume of 20 µl
1. Add the polymerase last
2. Mix by briefly vortexing the solution and spin 1 min at 13,000 rpm in a microfuge centrifuge.
3. Incubate the reaction mixture for 30 min at 22°C (or room temperature).
4. Incubate for 20 min at 75°C to inactivate the polymerase.
5. Spin 1 min at 13,000 rpm in a microfuge centrifuge.
The LIC prepared vector solution obtained in this way can be used directly in the annealing reaction (protocol 3). For longer term storage of the prepared vector it would be better to purify it further using for instance the MinElute PCR Purification Kit or Nucleotide Removal Kit (Qiagen). Take care that the final vector concentration is 10-20 ng/µl. The prepared vector can be stored at -20°C or lower.
2. Preparation of the insert
To create an insert with complementary overhangs to the EMBL-made LIC vectors the following primers have to be used:
Forward primer CAGGGCGCCATG-gene of interest
Reverse primer GACCCGACGCGGTTA-gene of interest (rev. comp.)
The forward primer should contain the complementary overhang (shown in red), the ATG start codon (underlined), and a long enough overlap with the gene of interest to give a melting temperature of 60°C or more.
The reverse primer should contain the complementary overhang (shown in red), one or more stop codons (e.g. TAA as shown here underlined) if no C-terminal tag is used, and a long enough overlap with the reverse complement strand of the gene of interest to give a melting temperature of 60°C or more.
2.1 PCR amplification of the insert
Materials Chemicals
200-µl PCR tubes agarose (electrophoresis grade)
1.5-ml microfuge tube dNTPs (10 mM each of dATP, dCTP, dGTP, dTTP)
ethidium bromide solution (10 mg/ml)
MinElute Gel Extraction Kit TBE buffer
Enzymes
Pfu DNA polymerase (2.5 U/µl)
10X Pfu polymerase buffer (supplied with the enzyme)
Mix in a 200-µl PCR tube:
5 µl 10X Pfu polymerase buffer
0.5 µl forward primer (100 pmol/µl)
0.5 µl reverse primer (100 pmol/µl)
* dNTPs (10 mM each)
0.5 µl DNA template
1 µl Pfu DNA polymerase (2.5 units/µl)
Add sterile water to a volume of 50 µl
* 20 ng for plasmid DNA
100 ng for genomic DNA
1. Add the polymerase last.
2. Mix by briefly vortexing the solution.
3. Perform the PCR as described below.
PCR protocol
Step Time Temperature Cycles
Denaturation 2 min 95°C 1
Denaturation 30 sec 95°C
Annealing 30 sec 56°C 30
Extension * 72°C
Extension 10 min 72°C 1
Hold 4°C 1
* use 1 min per kb for Pfu DNA polymerase
After the PCR it is important to remove the dNTPs completely from the reaction mixture. If the PCR template and the LIC vector have the same antibiotic resistance marker, the PCR product must be separated from the template. Both can be achieved by preparative agarose gel electrophoresis.
4. During the PCR prepare a 0.8% agarose gel.
Dissolve 0.4 g agarose in 50 ml TBE buffer by heating. After the solution has cooled down add 1-2 µl ethidium bromide solution and pour it into a prepared gel running chamber. After the gel has solidified fill the chamber with TBE buffer.
5. Add 10 µl 6X loading dye solution to the PCR product.
6. Load the sample on the agarose gel.
7. Run the gel for 1 hours at 100 V.
8. Analyze the gel on a UV lamp and cut out the band of the PCR product.
9. Purify the DNA form the gel pieces using the MinElute Gel Extraction Kit.
10. Elute the DNA in 30 µl elution buffer in a 1.5-ml microfuge tube.
2.2 T4 DNA treatment of the PCR product
In the next step, the PCR product is incubated with T4 DNA polymerase in the presence of dATP. Because of the 3'--> 5' activity of the polymerase the bases are removed from both 3'-ends until the first adenosine (A) residue is reached.
CAGGGCGCCATG...gene-of-interest...TAACCGCGTCGGGTC
GTCCCGCGGTAC...gene-of-interest...ATTGGCGCAGCCCAG
| T4 DNA polymerase + dATP
V
CAGGGCGCCATG...gene-of-interest...TAA
..........AC...gene-of-interest...ATTGGCGCAGCCCAG
For the annealing (protocol 3) 0.02 pmol of LIC prepared insert DNA is used. Below the T4 DNA polymerase treatment of the PCR product is set up with 0.2 pmol to produce enough material for 10 annealing reactions. This can be scaled up or down according to your own need.
The DNA concentration can be determined using the absorbance at 280 nm (assuming A280 = 1 is 50 ng/µl). To calculate the DNA concentration in pmol/µl apply:
number of base pairs x 0.65 = ng/pmol
For instance, for an insert of 1000 base pairs 0.2 pmol is equivalent to 130 ng.
Materials Chemicals
1.5-ml microfuge tubes dATP stock solution (100 mM)
DTT (100 mM)
100X BSA
Enzymes
T4 DNA polymerase
10X New England Biolabs buffer 3 (supplied with the polymerase)
Mix in a 1.5-ml microfuge tube:
2 µl 10X New England Biolabs buffer 3
0.2 pmol PCR product
0.5 µl dATP (100 mM)
1 µl DTT (100 mM)
0.2 µl 100X BSA
0.4 µl T4 DNA polymerase (?? units/µl)
Add sterile water to a volume of 20 µl
1. Add the polymerase last
2. Mix by briefly vortexing the solution and spin 1 min at 13,000 rpm in a microfuge centrifuge.
3. Incubate the reaction mixture for 30 min at 22°C (or room temperature).
4. Incubate for 20 min at 75°C to inactivate the polymerase.
5. Spin 1 min at 13,000 rpm in a microfuge centrifuge.
3. Annealing of the insert and the LIC vector
The complementary overhangs that are created in the vector (protocol 1) and insert (protocol 2) are long enough for the very specific, enzyme -free annealing of the two DNA.
CAGGGCGCCATG...gene-of-interest...TAA
..........AC...gene-of-interest...ATTGGCGCAGCCCAG
+
ATTTT.....................................CCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGT.......................................TGGTG
|
V
ATTTTCAGGGCGCCATG...gene-of-interest...TAACCGCGTCGGGTCACCAC
TAAAAGTCCCGCGGTAC...gene-of-interest...ATTGGCGCAGCCCAGTGGTG
The annealing reaction is set up as follows:
• 0.02 pmol of insert DNA.
• 25 - 50 ng* of LIC prepared vector DNA.
• The control ligation is carried out with sterile water instead of the insert.
* The amount of LIC prepared vector DNA needed depends on the size of the vector and the molar ration of vector to insert (normally 1:2 or 1:3 is used).
Example: LIC prepared pETM-11/LIC has a size of 5318 bp. With a 1:2 molar ratio you need 0.01 pmol vector in the annealing reaction. This is equivalent to 35 ng.
Materials Chemicals
1.5-ml microfuge tubes EDTA (25 mM)
Mix in a 1.5-ml microfuge tube:
1 µl LIC prepared vector DNA
2 µl T4 polymerase treated insert DNA
1. Incubate the annealing mixture for 5 min at 22°C (or room temperature).
2. Add 1 µl EDTA (25 mM).
3. Mix gently by stirring the solution with the tip.
4. Incubate for a further 5 min at 22°C (or room temperature).
The annealing is complete within 5 min of incubation but reactions can be incubated up to 1 h with equivalent results.
4. Transformation of the annealing product into E. coli DH5 competent cells
Materials
1.5-ml microfuge tubes
chemically competent E. coli DH5 cells
SOC medium
LB-agar plates containing 50 µg/ml kanamycin
1. Thaw the appropriate amount of competent DH5 cells on ice.
2. Transfer 1 µl of the annealing mixture to a 1.5-ml microfuge tube and incubate on ice for at least 5 min.
3. Add 50 µl aliquots of competent cells.
4. Incubate the tubes for 30 min on ice.
5. Heat shock the cells for 45 sec at 42°C.
6. Place the tubes immediately on ice and incubate for at least 2 min.
7. Add 200 µl SOC medium to each tube and incubate for 1 hour at 37°C in a shaker/incubator.
8. Spin for 1 min at 5,000 rpm in a microfuge centrifuge.
9. Remove 150 µl of supernatant and resuspend the cells in the remaining medium.
10. Plate out the cell suspension on a LB agar plate containing 50 µg/ml kanamycin.
11. Incubate the plates overnight at 37°C.
5. Identification of positive constructs
Materials Chemicals
1.5-ml microfuge tubes agarose (electrophoresis grade)
15-ml Falcon tubes 6X loading dye solution
LB medium dNTPs (10 mM each of dATP, dCTP, dGTP, dTTP)
Qiaprep Spin Miniprep Kit kanamycin (30 mg/ml)
Enzymes
restriction enzymes (here SmaI and XbaI)
Pfu DNA polymerase
10X restriction enzyme buffer (supplied with the enzymes)
10X Pfu DNA polymerase buffer (supplied with the enzyme)
5.1 Preparation of plasmid mini-preps
1. Pick 3 colonies from the positive plate and inoculate 3 x 4 ml LB medium containing 30 µg/ml kanamycin in 15-ml Falcon tubes.
The number of colonies picked depends on the ratio between the number of colonies on the positive and on the control plate (background). Usually the background is quite low and 3 colonies are sufficient but in some cases more colonies should be picked.
2. Incubate overnight at 37°C in a shaker/incubator.
3. Spin for 10 min at 4,000 rpm (table top centrifuge) and discard the supernatant.
4. Resuspend the pellets in the appropriate buffer to prepare plasmid mini-preps using the Qiaprep Spin Miniprep Kit (Qiagen).
To determine if the right size insert is present in the plasmid minipreps they can be analyzed using one or both of the following protocols: digestion analysis (protocol 5.2) and/or PCR analysis (protocol 5.3).
5.2 Digestion analysis of the plasmid minipreps
Since the LIC vector do not contain a multiple cloning site, you have to select 2 unique restriction sites in the vector backbone. For instance, with pETM-11/LIC the XbaI and SmaI sites could be used (see vector map in Appendix) but also other restriction sites are available.
Mix in a 1.5-ml microfuge tube:
2 µl 10X New England Biolabs buffer 4
0.2 µl 100X BSA
5 µl plasmid miniprep
1 µl XbaI (20 units/µl)
1 µl SmaI (20 units/µl)
Add sterile water to a volume of 20 µl
1. Add the restriction enzymes last
2. Mix by briefly vortexing the solution and spin 1 min at 13,000 rpm in a microfuge centrifuge.
3. Incubate the digestion mixture for 1-2 hours at 37°C.
4. In the meantime, prepare a 0.8% agarose gel.
Dissolve 0.4 g agarose in 50 ml TBE buffer by heating. After the solution has cooled down add 1-2 µl ethidium bromide solution and pour it into a prepared gel running chamber. After the gel has solidified fill the chamber with TBE buffer.
5. Add 4 µl 6X loading buffer to the samples.
6. Load the samples on the agarose gel.
7. Run the gel for 1 hours at 100 V.
8. Analyze the gel on a UV lamp.
5.3 PCR analysis of the plasmid minipreps
To determine if the right size insert is present in the plasmids mini-preps PCRs are performed using the forward and reverse primers for the gene of interest.
Mix in a 200-µl PCR tube:
5 µl 10X Pfu polymerase buffer
0.5 µl forward primer (100 pmol/µl)
0.5 µl reverse primer (100 pmol/µl)
1 µl dNTPs (10 mM each)
0.5 µl plasmid miniprep DNA
1 µl Pfu polymerase (2.5 units/µl)
Add sterile water to a volume of 50 µl
1. Add the polymerase last
2. Mix by briefly vortexing the solution.
3. Perform the PCR as described in "PCR experiments".
4. In the meantime, prepare a 0.8% agarose gel.
Dissolve 0.4 g agarose in 50 ml TBE buffer by heating. After the solution has cooled down add 1-2 µl ethidium bromide solution and pour it into a prepared gel running chamber. After the gel has solidified fill the chamber with TBE buffer.
5. Add 10 µl 6X loading buffer to the samples.
6. Load the 10-20 µl of the samples on the agarose gel.
7. Run the gel for 1 hours at 100 V.
8. Analyze the gel on a UV lamp.
Appendix 1
Materials
200-µl PCR tubes
1.5-ml microfuge tubes
15-ml Falcon tubes
SOC medium Invitrogen (15544-034)
chemically competent E. coli DH5 cells
MinElute PCR Purification Kit Qiagen (28006)
MinElute Gel Extraction Kit Qiagen (28606)
Qiaprep Spin Miniprep Kit Qiagen (27106)
Chemicals
agarose (electrophoresis grade) Invitrogen (15510-027)
dATP (100 mM) Roth (K035.1)
dNTPs (10 mM of dATP, dCTP, dGTP, dTTP)
dTTP (100 mM) Roth (K036.1)
DTT Roth (6908.2)
EDTA
ethidium bromide (10 mg/ml)
kanamycin sulfate Roth (T832.3)
6X loading dye solution Fermentas (R0611)
10X TBE buffer Roth (3061.2)
100X BSA New England Biolabs (B9001S)
Enzymes
BsaI New England Biolabs (R0535S)
Pfu DNA polymerase Fermentas (EP0502)
T4 DNA polymerase New England Biolabs (M0203S)
restriction enzymes New England Biolabs
Appendix 2
Available LIC vectors
Vector Promoter Selection Tag Protease cleavage site Origin
pETM-11/LIC T7/lac Kan N-His TEV pBR322
pETGB-1a/LIC T7/lac Kan N-His
N-GB1 TEV pBR322
pETZ2-1a/LIC T7/lac Kan N-His
N-Z-tag2 TEV pBR322
pETTrx-1a/LIC T7/lac Kan N-His
N-TrxA TEV pBR322
pETNus-1a/LIC T7/lac Kan N-His
N-NusA TEV pBR322
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