[求救] Cloning
看板Biotech (生命科學)作者alicialai (Fairy Monster)時間14年前 (2011/04/18 07:35)推噓6(6推 0噓 20→)留言26則, 11人參與討論串1/3 (看更多)
Hello, 大家早, 最近face到很annoy的問題,
我想了半天都想不出辦法來, 請大家幫忙解惑..
實驗步驟大致上是這樣,
因為我的final construct have to be in pDM4 vector (7kb)
但是因為這傢伙很胖+copy number低,
所以我選擇先在pBluescript上做construct.
我的final produc in pBluescript 是3kb (2個片段+一個antibiotic marker(Tet))
很不幸的, pBluescript也是3kb, 造成我無法用enzyme分開,
(other enzyme in pBS lab沒有, 新訂enzyme是最後的辦法)
所以我的處理是用respected enzyme切後直接purified做ligation,
之後用double antibiotics marker selection.
Tet(另外接進去的) and Cm which is the marker for pDM4,
這樣便可以殺掉self-ligation (指insert接回去pBS).
然後問題就來了!
Transform後隔天有colony, colony PCR (pDM4 primer) is positive.
但我inoculate to liquid LB之後他就不長了,
但同樣的colony我restreak on new plate(2 antibiotics)就長得很開心,
試了兩次都這樣... very annoyed!!!!!
想請問大家有沒有碰過類似的問題以及有沒有建議的方法呢?
感謝大家憂^__________________________^
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