📋 Workflow
Full workflow, products add-to-cart directly1
Colony DNA extraction
30 minPick single colony, extract genomic DNA by alkaline lysis or kit
💡 Tip Heat denaturation suits many Gram-negative
⚠️ Caution Complete lysis for intact DNA
🧪 Materials for this step (click to shop by spec/brand)
2
16S PCR amplification
2 hAmplify full-length or fragment of 16S gene with universal primers (e.g. 27F/1492R)
💡 Tip Universal primers cover broad taxa
⚠️ Caution Negative control against contamination
🧪 Materials for this step (click to shop by spec/brand)
3
Purify and sequence
Hours-1 dayVerify by gel, purify PCR product, obtain sequence by Sanger or high-throughput sequencing
💡 Tip Paired-direction sequencing improves accuracy
⚠️ Caution Purity affects sequencing quality
🧪 Materials for this step (click to shop by spec/brand)
4
Database comparison
30 minCompare sequence to databases with BLAST or EzBioCloud to determine species
💡 Tip ≥99% similarity identifies to species
⚠️ Caution Low similarity needs phenotypic confirmation
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Poor DNA purity fails PCR
- ② Primer contamination gives false sequence
- ③ Poor sequence quality hampers alignment
- ④ Low similarity cannot determine species
- ⑤ Incomplete database causes misidentification
❓ FAQ
+What similarity defines species?
≥98.7% 16S similarity usually indicates same species; higher is more reliable.
+Can 16S distinguish all close relatives?
Some close species need additional methods like whole-genome or biochemistry.
+Full length or fragment?
Full length gives more info but needs long reads; hypervariable regions suit community profiling.