Re: [求救] TAIL PCR
作I-PCR之前
請用轉殖株抗性確認3/4抗 或是 southern 偵測 copy numbers
確認為single copy 再去作
固定(T-DNA的 LB 或 RB)一端 去設計實驗
1. genomic DNA isolation
2. G-DNA Restriction Enzyme(RE) digestion
***U may need to try multiple REs (it depends...)***
3. to remove RE by PCI, column or to stop the RE activity by chemicals
4. digested-DNA self-ligation >> to generate a circular form of DNA
5. 1st PCR: to amplify the flanking sequences by using the 1st pair of
"out-going" primers
第一刀 (left border 外面) 第二刀 (T-DNA上,引子外)
1#(RE) 2#(RE)
(未知序列) LB T-DNA(已知序列) RB
----------------============================================================---
2nd primers <--- ----->
1st primers <---- ------->
6. To confirm the result of the 1st PCR products by using the 2nd pair of
"out-going" primers
**** Make sure u have tried many TM conditions in the 1st & 2nd PCR ****
7. gel extract some target bands
8. send to a comerical agency
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