📋 Workflow
Full workflow, products add-to-cart directly1
Primer design and PCR
2 hDesign primers with restriction sites or homology arms, PCR-amplify target gene
💡 Tip Similar Tm, avoid secondary structures
⚠️ Caution Use high-fidelity polymerase to reduce mutations
🧪 Materials for this step (click to shop by spec/brand)
2
Vector and insert preparation
2 hDouble-digest vector and PCR product with restriction enzymes, gel-purify target fragments
💡 Tip Gel purification removes residual enzyme/template
⚠️ Caution Complete digestion prevents self-ligation
🧪 Materials for this step (click to shop by spec/brand)
3
Ligation
1 hMix vector:insert at molar ratio, add T4 ligase to ligate, or use recombinational cloning
💡 Tip Optimize insert:vector molar ratio
⚠️ Caution Include self-ligation control
🧪 Materials for this step (click to shop by spec/brand)
4
Transformation and screening
1 dayTransform competent cells by heat-shock/electroporation, screen on antibiotic plates, verify clones by PCR/sequencing
💡 Tip Include transformation control for efficiency
⚠️ Caution Sequence clones to confirm correct insert
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Incomplete digestion causes self-ligation false positives
- ② PCR-introduced mutations affect protein expression
- ③ Wrong insert orientation prevents expression
- ④ Low transformation efficiency yields no positive clones
- ⑤ No sequencing verification allows sequence errors
❓ FAQ
+How to confirm insert orientation?
Verify by directional PCR, restriction mapping or final sequencing.
+Recombinational vs restriction cloning?
Recombinational is higher-throughput and enzyme-free; traditional is more flexible.
+How to reduce PCR mutations?
Use high-fidelity polymerase, fewer cycles, or synthesized gBlocks.