我的lab 買了一株還特定clone 之菌
該 vector 為 pSin-EF2-Pur
遽我所知此載體將用來做感染細胞做病毒用
買了的菌株是 STBL3
查了一下這株菌
含有endonuclease 1 (endA1+) :
Reference:
摘錄自invitrogen 的 Kit:
pLenti6/V5 Directional TOPOR Cloning Kit
http://tools.invitrogen.com/content/sfs/manuals/plenti6v5dtopo_man.pdf
Stbl3? E. coli is wild type for endonuclease 1 (endA1+). When performing
plasmid DNA isolation with commercially available kits, ensure that Solution
I of the Lysis buffer (often called Resuspension Buffer) contains 10 mM
EDTA to inactivate the endonuclease to avoid DNA nicking and vector
degradation.
當我養菌, 並用 geneid 的 mini kit 抽載體時,
載體若泡在限制梅的Buffer就會被切爛
後來我聽說只要把載體轉染至 DH5alpha
就不會有這樣的現象
Reference:
Problems with STBL3 cells - Digesting DNA from STBL3 cells
http://www.protocol-online.org/biology-forums/posts/11640.html
想請問過來人的經驗:
1. STBL3 這株菌有特殊用途嗎? 為甚麼要用這株菌?
2. 我要操作vector pSin-EF2-Puro 以便將來製作病毒感染細胞用
可以轉染到DH5alpha 來操作就好嗎?
感謝~~
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