GenieClone Ultra DNA Assembly Kit (MORV0089)

Components

Self-prepared materials: PCR templates, primers, linearised vector, a high-fidelity DNA polymerase, chemically competent cloning cells, ddH2O, PCR tubes and a thermal cycler.

Storage

Store at -30 ~ -15°C.

1. Linearise the vector
   • Choose a cloning site free of repeats, with 40%–60% GC in the 20 bp up- and downstream of the site.
   • Restriction digestion: double digestion is preferred over single digestion for complete linearisation. Extend digestion time to reduce circular plasmid carry-over and false-positive background.
   • Inverse PCR: use a high-fidelity DNA polymerase and 0.1–1 ng circular or pre-linearised plasmid as template in a 50 μl reaction. If the template is circular, digest the product with DpnI and gel-purify it.
2. Prepare the inserts
   • Inserts can be amplified with Taq or high-fidelity polymerases; any A-overhang is removed during recombination. A high-fidelity polymerase is recommended to minimise mutations.
   • Single fragment: add a 15–20 bp vector-end homologous sequence (excluding restriction sites) to the 5' end of each primer. Forward primer = 5' - upstream vector-end homology + restriction site (optional) + gene-specific forward sequence - 3'; reverse primer = 5' - downstream vector-end homology + restriction site (optional) + gene-specific reverse sequence - 3'.
   • Multiple fragments: design the outermost primers as above. Between adjacent inserts, add 15–20 bp of homology by extending the downstream fragment with the 3' end of the upstream fragment, the upstream fragment with the 5' end of the downstream fragment, or splitting the overlap between both.
   • Gene-specific sequences: Tm 60 ~ 65°C (exclude homology arms when calculating Tm). Homology arms: 40%–60% GC. PAGE-purify primers longer than 40 bp.
3. Quantify DNA and calculate input
   • For gel-purified DNA without extra bands or smears, absorbance readings are reliable only at A260/A280 1.8–2.0. Readings below 10 ng/μl may differ between instruments.
   • Single fragment (vector:insert 1:2): vector = [0.02 × bp] ng (0.03 pmol); insert = [0.04 × bp] ng (0.06 pmol). If the insert is longer than the vector, invert the calculation.
   • Multiple fragments (1:1): vector and each insert = [0.02 × bp] ng (0.03 pmol).
   • Use 10–100 ng of each; if the calculated value falls outside this range, use the nearest limit. For single-fragment reactions, unpurified DNA with no extra bands may be used at ≤2 μl total (1/5 of the reaction), though efficiency will be lower.
4. Set up the recombination on ice

^a X/Y = calculated vector/insert amounts. Dilute DNA so that each volume is at least 1 μl.
^b Checks for circular plasmid carry-over in the linearised vector.
^c Recommended when the PCR template is a circular plasmid with the same antibiotic resistance as the vector.
^d Uses the supplied control insert and vector to rule out problems with other materials or handling.

5. Run the recombination
   • Mix gently by pipetting (do not vortex) and spin down.
   • Incubate at 50°C for 5–15 min (single fragment) or 15–30 min (multiple fragments; use 30 min for >5 inserts), then chill immediately at 4°C or on ice. A thermal cycler is recommended.
   • If total vector and insert volume exceeds 5 μl, reduce input (within 0.003–0.25 pmol) rather than extend the reaction time.
   • The product can be stored at -20°C for one week.
6. Transform
   • Thaw cloning competent cells on ice. Add 5–10 μl recombination product to 100 μl cells (≤1/10 of the cell volume), flick to mix (do not vortex) and keep on ice for 30 min.
   • Heat-shock at 42°C for 30 sec, then place on ice for 2–3 min.
   • Add 900 μl SOC or LB medium (no antibiotic) and shake at 37°C, 200–250 rpm for 1 h.
   • Centrifuge at 5,000 rpm (2,500 × g) for 5 min, discard 900 μl supernatant, resuspend and spread on a pre-warmed selective LB plate.
   • Incubate at 37°C for 12–16 h.
7. Identify recombinants
   • Colony PCR: resuspend colonies in 10 μl ddH₂O as template, using at least one vector primer.
   • Restriction analysis: culture colonies overnight in selective medium, extract plasmid and digest.
   • Sequencing with vector primers.

Q1: How should I design primers?

• Each primer has three parts: a 15–20 bp homology arm (excluding restriction sites, 40%–60% GC), an optional restriction site and the gene-specific sequence. Exclude the homology arm when calculating Tm.

Q2: Why are there few or no colonies?

• Check that primers carry 15–20 bp homology arms with 40%–60% GC; use the recommended vector and insert amounts and ratio; keep unpurified DNA ≤2 μl (1/5 of the reaction) or gel-purify and dissolve in ddH₂O (pH 8.0); and use cloning (not expression) competent cells, such as DH5α, with efficiency >10⁸ cfu/μg. Keep the transformation volume ≤1/10 of the cell volume.

Q3: Why do most clones lack the insert or carry the wrong one?

• Non-specific PCR products may be present: optimise the PCR, gel-purify the product and screen more colonies. The vector may be incompletely linearised: check with the negative control, use more enzyme or longer digestion and gel-purify. A circular plasmid template with the same resistance may be carried over: digest the PCR product with DpnI or gel-purify it.

Q4: Why is there no target band in colony PCR?

• Use vector universal primers, or at least one universal primer. If neither target nor empty-vector bands appear, optimise the PCR, use extracted plasmid as template or check by restriction digestion. If only the empty-vector band appears, recombination failed because vector linearisation was incomplete; optimise the digestion.

Q5: How can I check competent cell efficiency?

• Transform 0.1 ng of plasmid and plate 1/10 of the culture. About 1,000 colonies corresponds to 10⁸ cfu/μg.