[求救] Ligation
Insert 大小為1.5k 已放入TA裡
Vector pcDNA3.1(+) 大小為5.5k
比較麻煩的是 Insert和Vector可以用的cut site有點複雜
→
---+-----+-[ gene ]-+-----+--- Insert-pGEM-T easy
EcoRI BamHI Hind3 EcoRI
→
------------+----+------------ Vector pcDNA3.1(+)
BamHI EcoRI
(G/GATCC)--17nt--(G/AATTC)
我以BamHI 和EcoRI作為連結處
Digestion protocol為
Insert(10ug~30ug) BamHI先切 1hr 再加入EcoRI 並補滿buf 再切2hr
^此步有先取1ul出來跑膠 確定有切完
Vector(10ug~30ug) BamHI+EcoRI 同時切3hr
gel extraction >
ligation (molar ratio=1:1, 1:3, 3:1) RT 3hr >
transformation
positive control丟約200ng 長滿長片
ligation p/c 用vector切一刀 丟40ng去ligation 約有500顆
但實驗組永遠都只有1~2顆的colonies
而且抽出來size都不正確
想請問這些實驗步驟中有沒有什麼問題?
會不會是Vector的兩個cut size太近 時間太短?
還是可能是Ligation步驟問題?
謝謝各位:)
祝大家實驗順利
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◆ From: 140.112.125.32
※ 編輯: ararthur 來自: 140.112.125.32 (09/13 12:37)
推
09/13 13:43, , 1F
09/13 13:43, 1F
請問這裡是說用切兩刀的vector 還是切一刀的?
切一刀的有做過 約500顆
→
09/13 13:45, , 2F
09/13 13:45, 2F
→
09/13 13:46, , 3F
09/13 13:46, 3F
→
09/13 13:47, , 4F
09/13 13:47, 4F
→
09/13 13:47, , 5F
09/13 13:47, 5F
→
09/13 13:48, , 6F
09/13 13:48, 6F
→
09/13 13:48, , 7F
09/13 13:48, 7F
→
09/13 13:49, , 8F
09/13 13:49, 8F
※ 編輯: ararthur 來自: 140.112.125.32 (09/13 14:48)
推
09/13 19:55, , 9F
09/13 19:55, 9F
→
09/13 19:55, , 10F
09/13 19:55, 10F
推
09/14 19:34, , 11F
09/14 19:34, 11F
→
09/15 00:11, , 12F
09/15 00:11, 12F
→
09/15 06:42, , 13F
09/15 06:42, 13F
→
09/15 09:12, , 14F
09/15 09:12, 14F
→
09/15 09:12, , 15F
09/15 09:12, 15F
※ 編輯: ararthur 來自: 111.249.167.145 (09/15 09:13)
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