SLIC Cloning
1. PCR
Amplify the vector(s) and your gene of interest with the LP1/LP2 primers and gene specific SLIC primers (designed with ~20 bp homology to vector), respectively. Use a high fidelity polymerase likePfu, Phusion, etc. in order to avoid mutations. The PCR products are purified by PCR purification columns. Quantify the inserts.
2. T4 DNA polymerase treatment
Take 1 µg of the vector and 1 µg of the inserts treat separately with 0.5 U of T4 DNA polymerase in T4 buffer (NEB) plus BSA or 10 X CutSmart Buffer (which does not require BSA) in a 20 μl reaction at room temperature for 30 minutes.Stop the reaction by adding 1/10 volume of 10 mM dCTP and leave on ice. Both the gel-purified vector and insert must be treated separately with T4 DNA polymerase.
| 15.6 µl | vector or insert | |
| 4 µl | 5x buffer | |
| 0.2 µl | T4 DNA polymerase (5U/µl) | RT incubation for 30 min |
| 0.2 µl | 10 mg/ml BSA | |
| 20 µl | final vol. |
Stop the reaction with 2 µl 10 mM dCTP and place on ice.
3. Annealing
Set up a 10 µl annealing reaction using 1:1 insert to vector ratio with 100-150 ng of a the vector, 1x ligation buffer (NEB), appropriate amount of insert (molar ratio insert/vector between 1/1 and 2/1) and water. Incubate at 37°C for 30minutes. Leave on ice or store in -20°C. Note: We saw a clear correlation of the size of the fragments and the number of colonies after transformation (fewer colonies with increasing size), so the amount of insert has to be adjusted according to its size in order to have similar molar ratios. Annealing reaction (different ratios of insert to vector can be tested)
| insert | 3 µl |
| vector | 3 µl |
| 10X ligation buffer | 1 µl |
| RecA protein | 20 ng |
| H2O | 3 µl |
| 10 µl final vol. | |
4. Transformation
3 µl annealing reaction is added to 50 µl Top10 cells, electroporated, then 300 µl SOC added, shaken at 37°C for 30-45 min and then everything plated.
Direct Cloning of PCR Product ==
Purify PCR product (combined), elute with 20 ul H2O.
Add 2 ul 10 X CutSmart buffer
Add .75 U (0.25 µL) T4 DNA polymerase,
Incubate at RT for 2.5 to 10 min
Add place on ice for 10 min.
Transform Top10
SLIC using T4P mutant Y320A
[edit]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343910/ 200 ng of purified PCR products (Vector) in 20 μL in T4 DNA pol buffer
Add 1 μL of 4 μM (0.4 mg/mL) stock solution of E2 enzyme
Incubate for 30 min h at 37 °C.
Heat to 72 °C for 20 min
Cool on bench for 5 min
Put on ice
Use 5 ul ~ 20 ul for transformation
Primers: 15 nt overlap, melting temperature of the overlap is at least 45 °C, preferably 50–55 °C.
Properties of DNA Polymerases
| 5'—>3' Exonuclease | 3'—>5' Exonuclease | Error Rate(x10-6) | Strand Displacement | Nick Translation | Thermal Stability | Km dNTPs | Km DNA | Extend RNA Primer | Extension from Nick | Primary Applications | |
| Klenow Fragment 3´→ 5´ exo– |
– | – | 100 | +++ | – | – | + | + | Labeling | ||
| Klenow Fragment DNA Polymerase I |
– | ++ | 18 | ++ | – | – | 2 µM | + | + | Polishing Ends | |
| Phusion High-Fidelity DNA Polymerase* |
– | ++++ | 0.44 | – | – | +++ | – | + | PCR (high fidelity) | ||
| T4 DNA Polymerase | – | ++++ | <1 | – | – | – | 2 µM | + | – | Polishing Ends, 2nd Strand Synthesis | |
| T7 DNA Polymerase | – | ++++ | 15 | – | – | – | 18 µM | 18 nM | + | – | Site Directed Mutagenesis</tbody> |
| 3'->5' Proofreading | Strand Displacement | Primary Applications | |
| Mesophilic DNA Polymerases | |||
| phi29 DNA Polymerase | ++++ | +++++ | Strand Displacement Applications |
| T4 DNA Polymerase | +++++ | - | Polishing Ends, 2nd Strand Synthesis |
| DNA Polymerase I | ++ | -* | Nick Translation |
| DNA Polymerase I, Klenow Fragment | ++ | ++ | Polishing Ends |
| Klenow Fragment (3' -> 5' exo-) | - | +++ | Labeling |
| T7 DNA Polymerase (unmodified) | ++++ | - | Site Directed Mutagenesis |
Properties of Exonucleases and Endonucleases
This table is intended to be used as a guideline. Not all reported activities and properties for each exonuclease or endonuclease are listed. The amount of enzyme, substrate and time of incubation can have a dramatic effect upon the desired outcome of the experiment.
| Enzyme | polarity | DNA substrate | Activity without 5' phosphate |
Able to Initiate on DNA with1 | Partial Digestion ss Extension2 |
Products Produced3 | End- labeled |
Phospho-rothioate cleavage5 | Units for 90% digestion of 2 µM oligos6 | ||||
| ss | ds | 5'ext | 3'ext | blunt | nick | ||||||||
| Lambda Exo | 5'->3' | +/- | + | +/-7 | +/- | + | + | - | 3' | ssDNA, dNMP | + | - | 2 |
| T7 Exo | 5'->3' | +/- | + | + | +/- | + | + | + | 3' | ssDNA, dNMP, dinucleotide8 | + | - | 10 |
| Exo III | 3'->5' | +/- | + | + | + | +/- | + | + | 5' | ssDNA, dNMP | + | - | 10 |
| RecJf | 5'->3' | + | - | + | +/-9 | - | +/-10 | - | NA | dNMP | +/- | - | >1500 |
| Exo I | 3'->5' | + | - | + | - | +/-9 | +/-10 | NR | NA | dNMP, dinucleotide11 | + | - | 20 |
| Exo T | 3'->5' | + | - | + | - | +12 | +/-10 | NR | NA | dNMP | + | - | >100 |
| BAL-31 | 3'->5', endo13 |
+ | + | + | + | + | + | + | NA | dsDNA, dNMP | NA | NA | NR |
| Mung Bean |
endo | + | - | + | + | + | - | - | NA | ssDNA, dsDNA | NA | NA | 10 |
| DNase I | endo | + | + | NA | NA | NA | NA | NA | NA | ssDNA, dsDNA oligonucleotides, di and trinucleotides | NA | NA | 0.1 |
| Micrococcal |
endo | + | + | NA | NA | NA | NA | NA | NA | ssDNA, dsDNA 3´ monophospho and diphosphonucleotides14 | NA | NA | NR |
| T7 Endo I15 | endo | - | + | NA | NA | NA | NA | +/- | NA | dsDNA | NA | NA | NA |
| T5 Exo | 5'->3' | + | + | + | + | + | + | + | NA | dNMP to 6 mer | + | + | 2 |
Table Legend:
| + : | activity, preferred substrate |
| - : | no significant activity |
| +/- : | activity greatly reduced relative to preferred substrate |
| NR : | not reported |
| NA : | not applicable |
| ss : | single-stranded |
| ds : | double-stranded |
| ext : | extension |
| dNMP : | deoxyribonucleoside monophosphate |
Footnotes:
- The ability to act on short extensions and blunt ends distinguishes these enzymes; such ends are conveniently generated by restriction digestion. The 5´ and 3´ extensions tested were 4 nt in length.
- Partial digestion of dsDNA by Lambda Exonuclease, T7 Exonuclease and Exonuclease III will produce dsDNA products with ss extensions. Complete digestion produces ssDNA as a product.
- Complete hydrolysis of the preferred substrate will generate the listed products.
- The ability of an exonuclease to initiate on the end of the preferred DNA substrate (ss or ds) containing a fluorescein group linked to either the 5´ or 3´ end. Phosphoramidite chemistry was used to synthesize oligonucleotides with FAM groups. The 5´ FAM was added to the oligonucleotide as a [(3´,6´-dipivaloylfluoresceinyl)-6-carboxamidohexyl]-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite while the 3´ FAM oligonucleotides were synthesized using the 1-dimethoxytrityloxy-3-[O-(N-carboxy-(di-O-pivaloyl-fluorescein)-3-aminopropyl)]-propyl-2-O-succinoyl-long chain alkylamino-CPG support.
- The Sp stereoisomer of the phosphorothioate linkage is known to greatly inhibit cleavage of many nucleases while the Rp stereoisomer shows either no or less inhibition. Synthetic oligonucleotides containing approximately an equal ratio of both isomers necessitates the use of multiple phosphorothioates to block cleavage. The presence of 6 consecutive phosphorothioates on oligonucleotides of the preferred substrate blocked all exonucleases effectively (5% or less degradation) except T7 exonuclease which had approximately 10% cleavage. Analysis of exonucleases with oligonucleotides having either one or two consecutive phosphorothioates revealed significant degradation (not shown).
- The amount of enzyme in units to cleave greater than 90% of 2 µM 35-mer oligonucleotide (s) of the preferred substrate (ss or dsDNA) in a 10 µl volume using the unit digest reaction conditions. All enzymes tested except for Exo T and RecJf could effectively cleave the substrate to completion.
- Lambda exonuclease has a strong preference for initiating degradation on dsDNA containing a 5´ phosphate. Thus, if linear dsDNA has a 5´ phosphate at one end and lacks a 5´ phosphate on the other end, then lambda exonuclease will preferentially degrade the DNA from the phosphorylated end.
- It has been reported that the initial first product hydrolyzed from dsDNA by T7 Exonuclease is a dinucleotide. Subsequent hydrolytic cleavage releases dNMP.
- RecJf is not suitable for making 5´ extensions blunt. Exo I is not suitable for making 3´ extensions blunt. Both RecJf and Exo I require longer length ssDNA extensions to initiate than those generated by restriction enzymes.
- Depending upon the DNA sequence and amount of exonuclease, RecJf, Exo I and Exo T may remove a few nucleotides from flush termini
- Exo I releases dNMP from ssDNA, except at the last hydrolytic step where a dinucleotide is produced.
- Exo T can be used to make 3´ extensions blunt: however, it yields 2–4 fold fewer ligatable blunt ends when compared to Klenow polymerase plus dNTP on a test substrate.
- BAL31 has been reported as having both ss endonuclease activity as well as 3´→5´ ds exonuclease activity. Thus, any linear DNA is a substrate for BAL31.
- Products of Micrococcal Nuclease degradation have 3´ phosphates.
- T7 endonuclease recognizes and cleaves non-perfectly matched DNA, cruciform DNA, Holliday structures or junctions. It will act more slowly on nicked dsDNA.
| Enzyme |
Description |
Application/note |
Source |
Reaction Buffer |
| T7 DNA Ligase | T7 DNA Ligase catalyzes the formation of a phosphodiester bond between a 5′ phosphate and a 3′ hydroxyl termini in duplex DNA. The enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA | In the absence of high concentration crowding agents (eg 20-30% PEG 6-8000), the specific activity of T7 DNA Ligase is reduced by 1000-fold on blunt-ended fragments (1, 2) | A recombinant E. coli strain carrying the T7 DNA Ligase gene | 2X Rapid Ligation Buffer (B1010): 132mM Tris-HCI 20 mM MgCl2 2 mM DTT 2 mM ATP 15% PEG 6000 pH 7.6 @ 25°C |
| T4 DNA Polymerase |
Extreme fidelity (6)
T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. This enzyme has a 3´→ 5´ exonuclease activity which is much more active than that found in DNA Polymerase I. Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5´→ 3´ exonuclease function |
3´ overhang removal to form blunt ends (1,2). 5´overhang fill-in to form blunt ends (1,2). Single strand deletion subcloning (3). Second strand synthesis in site-directed mutagenesis (4). Probe labeling using replacement synthesis (1,2). |
Purified from a strain of E. coli that carries the T4 DNA Polymerase gene. |
1X NEBuffer 2: 10 mM Tris-HCl 50 mM NaCl 10 mM MgCl2 1 mM Dithiothreitol pH 7.9 @ 25°C |
| T5 Exonuclease |
T5 Exonuclease degrades DNA in the 5´ to 3´ direction (1). T5 Exonuclease is able to initiate nucleotide removal from the 5´ termini or at gaps and nicks of linear or circular dsDNA (1). However, the enzyme does not degrade supercoiled dsDNA (2). The ssDNA endonuclease activity has been shown to be suppressed by lowering the concentration of Mg2+ to less than 1 mM (1). This protein is the product of T5 phage D15 gene (3) | Degradation of linear ssDNA, dsDNA or nicked plasmid DNA and reserving supercoiled plasmid DNA | An E. coli strain that carries a plasmid with the T5 phage D15 gene |
1X NEBuffer 4: 20 mM Tris-acetate 50 mM potassium acetate 10 mM Magnesium Acetate 1 mM Dithiothreitol pH 7.9 @ 25°C |
| Lambda Exonuclease |
A highly processive enzyme that acts in the 5´ to 3´ direction, catalyzing the removal of 5´ mononucleotides from duplex DNA. The preferred substrate is 5´-phosphorylated double stranded DNA, although it will also degrade single-stranded and non-phosphorylated substrates at a greatly reduced rate. Lambda Exonuclease is unable to initiate DNA digestion at nicks or gaps (1) |
|
A genetic fusion of the E. coli Lambda Exonuclease gene with the gene encoding maltose binding protein (MBP). Following affinity chromatography, Lambda Exonuclease is cleaved from the fusion construct and purified away from MBP |
1X Lambda Exonuclease Reaction Buffer: 67 mM Glycine-KOH 2.5 mM MgCl2 50 µg/ml BSA pH 9.4 @ 25°C |
Competent cells
[edit]TOP10 (Invitrogen)
Inoculate TOP10 in 20 mL LB-medium in a 100 mL sterile Erlenmeyer flask
Incubate overnight at 37°C, 300 r.p.m.
Use the pre-culture to inoculate four 500 mL sterile Erlenmeyer flasks, each containing 250 mL LB-medium
Incubated at 37°C, 250 r.p.m, until OD600 = 0.5–0.6 is reached.
Cool down cultures on ice for 5 min
Spin down cells in autoclave tube at 4°C for 5 min at 4,000 g.
Resuspend cell pellets carefully in a total volume of 80 mL of freshly prepared (from stock solutions) ice-cold TFB1 buffer
34.4 mL ddH2O, 8 mL 300 mM potassium acetate pH 6.0 (adjusted with KOH), 1.6 mL 500 mM CaCl2, 8 mL 500 mM MnCl2, 16 mL 500 mM RbCl, 12 mL glycerol, sterile filtered
Spin cells were for 5 min at 4°C at 2,000 g and discard the supernatant.
Resuspend cells in a total volume of 8 mL of freshly prepared (from stock solutions) ice-cold TFB2 buffer
4.72 mL ddH2O, 0.8 mL 100 mM MOPS pH 7.0, 1.2 mL 500 mM CaCl2, 80 μL 1M NaCl, 1.2 mL glycerol, sterile filtered
Incubate on ice for 15 min.
Transfer aliquots of 25 μL to sterile 1.5 mL Eppendorf tubes aand store at -80°C for further use