原核生物之菌種鑑定PCR法(Prokaryote species identification method by PCR)

Target: 16s rRNA gene
Primer E8F: AGA GTT TGA TCC TGG CTC AG
Primer U1510R: CGG TTA CCT TGT TAC GAC TT
Expected product size: ~1500 bps

For Taq DNA polymerare:
25 μl/reaction
10 x Buffer ------------------------ 2.5 μl
dNTP (2.5 mM) ---------------------2 μl
Primer E8F (5 μM) ------------------1 μl
Primer U1510R (5 μM) -------------1 μl
Taq polymerase (2 U/μl) ----------0.2 μl
ddH2O ----------------------------18.3 μl

如果使用DNA作為模板(添加不超過2 μl)之條件
95oC 95oC 50oC 72oC 72oC 12oC
40 sec 30 sec 30 sec 1 min 30 sec 7 min ∞
└────── x 35 ───────┘

如果使用菌落作為模板 (可用微量吸管點一下菌落，然後在PCR管壁上轉個10圈)之條件
95oC 95 oC 50oC 72oC 72oC 12oC
5 min 30 sec 30 sec 1 min 30 sec 7 min ∞
└────── x 35 ───────┘

After electrophoresis, if the PCR product is a single banding, send for Sanger sequencing and BLAST the result on NCBI website.