This inversed PCR protocol is modified from minimos inverse PCR protocol

Only used for the introgression strains with genomic insertion of pzz31 vector.

1. Isolation of genomic DNA

We use the qiagen kit for DNA extraction.

2. DNA digest:

Digest 150 ng genomic DNA with HindIII-HF, 37 C for 3h.

25 ul system

• DNA : 150 ng
• HindIII enzyme: 1.0 ul
• Cutter smart buffer (10X): 2.5 ul
• H2O: add to 25 ul

3. Ligation

2h at room temperature or 16 C overnight.

25 ul system

• Digested DNA from step 2, 2.5ul (10x dilution)
• buffer (10x), 2.5 ul
• T4 ligase, 1ul
• H2O, add to 25ul

4. First round PCR

25 ul system

PCR setting

-30 cycles-
--95,30s--
--64,20s--
--72,3min--

• Ligation mix from step 3, 2.5 ul (10x dilution)
• Primer GFPin (F and R mixture), 2.5 ul
• dNTPs, 2.5 ul
• Phusion 5X GC buffer, 5 ul
• Phusion Polymerase, 0.25 ul
• H2O, 12.25 ul

5. Second round PCR

25 ul system

PCR setting

-30 cycles-
--95 C,30s--
--64 C,20s--
--72 C,3min--

• 100X dilution PCR product from step 4, 2.5 ul
• Primer GFPout (F and R mixture), 2.5 ul
• dNTPs, 2.5 ul
• Phusion 5X GC buffer, 5 ul
• Phusion Polymerase, 0.25 ul
• H2O, 12.25 ul