作者davideason (Davideason)
看板Biotech
标题Re: [求救] TAIL PCR
时间Mon Oct 20 16:54:47 2008
作I-PCR之前
请用转殖株抗性确认3/4抗 或是 southern 侦测 copy numbers
确认为single copy 再去作
固定(T-DNA的 LB 或 RB)一端 去设计实验
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
--
◢ p
╭▲╮ q
◣ 你也想加入肾斗士的行列吗!!
◢██ ◥◥▃██▃◤◤ ██◣ 三聚氰胺能让你的小宇宙爆发
◢███◣ ◥◥◥▼◤◤◤ ◢███◣ 也能让你的潜能无限结石无穷
███◣◣ ◥@ ⊙ ⊙ @◤ ◢◢███▌
今天你相信我明天你会相信他
◥███◣◣◣ ◥ 皿 ◤ ◢◢◢███◤ 请认明卫生鼠合格字样2.5ppm
◥ ██◣◣◣◣ ◢█ █◣ ◢◢◢◢██ ◤ψQSWEET
--
※ 发信站: 批踢踢实业坊(ptt.cc)
◆ From: 118.171.10.138
1F：推 flowwater:谢谢你帮了这麽多忙 最後还有一个小小低请求 10/20 18:50
2F：→ flowwater:可以给我protocol的文献出处吗? 拜托~~谢谢 10/20 18:51
3F：→ davideason:时间久远 我忘了~ 你网路查一下巴 10/20 20:08