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rbc ta cloning vector 在 [求救] TA cloning錯誤insert請教- 看板Biotech - 批踢踢實業坊 的八卦


作者ernielwl (ernielwl)
看板Biotech
標題[求救] TA cloning錯誤insert請教
時間Tue Oct 3 14:04:59 2017


請教經驗豐富的各位
做TA cloning會常常挑到錯誤的insert嗎
小弟使用RBC TA cloning kit
inserts 大小為1.5k，PCR產物經過gel extraction 純化
按照protocol 操作，ligation,transform結果都很好
但是藍白篩挑的白色菌落常常有錯誤insert

(藍白篩效果也沒有不好)



我們lab是從藍白篩挑白色菌起來，先在固態ampicillin LB做一次純培養，再到液體ampi
cillin LB隔夜
之後用Geneaid mini plasmid kit抽質體
質體由HindIII在37度切30min後跑電泳確認inserts
附上vector map

確定HindIII有兩個site可以切下中間的insert

然後切過的質體，常常在500bp,1kb跑出兩個產物，如附圖，34567跑道，第8跑道先前做
好的，正確的對照組
(marker使用100bp plus Dna ladder)


一開始老師以為那可能是insert裡面有HindIII切位，500bp+1k剛好是1.5kb
但是我在基因序列上分析過並沒有HindIII切位
還是有送其中一個sample去定序，
回來的結果經過blastn得到的都是一些vector


小弟實在不了解，會產生這些錯誤insert的原因是什麼? 是TA vector自己產生奇怪的自
接嗎？





--
※ 發信站: 批踢踢實業坊(ptt.cc), 來自: 101.8.211.165
※ 文章網址: https://www.ptt.cc/bbs/Biotech/M.1507010702.A.AFB.html
※ 編輯: ernielwl (101.8.211.165), 10/03/2017 14:07:02
※ 編輯: ernielwl (101.8.211.165), 10/03/2017 14:10:11
→ xxtomnyxx: 我會用 M13 forward 和 M13 reverse 去做 colony PCR10/03 15:00
→ xxtomnyxx: 有可能是 vector 有 contamination10/03 15:02
我們lab不習慣從colony PCR，boss說會有偽陽性，最後質體還是會抽，用質體出來跑是
最準的
然後我用M13 forward 分別我Insert的 forward, reverse primer去PCR確認，不然M13 F
,R跑出來大小還是一樣1.5kb分不清處
※ 編輯: ernielwl (101.8.211.165), 10/03/2017 15:08:39
→ blence: 約1.5k這一段,跟起始作為PCR template也沒有關係嗎？10/03 18:15
前陣子做insert只有400~700bp的時候也會出現這段

這次剛好clone 1.5kb的產物所以有一起去定序，真的一點關係都沒有
非常詭異......而且跟vector的序列去alignment也不是100%對到其中一段

推 rebirth: 先colony PCR screening,挑出對的size insert10/03 19:03
→ rebirth: 再抽plasmid做 RE check10/03 19:04
我們跳過colony PCR直接繼續下去RE check，結果來說其實一樣，做起來習慣就好
※ 編輯: ernielwl (175.96.64.191), 10/03/2017 19:25:51
推 lail: 會耶，之前做TA多少會跳到奇怪的clone(定序結果)，所以除非10/03 20:46
→ lail: 不得已，不然不太用TA cloning10/03 20:46
推 milkpa: lane2/345/67/8 ，總共有四種pattern，各拿一個去定序 10/03 20:59
→ xxtomnyxx: 我會猜你們的 TA vector 可能混到其他不知名的 vector10/03 21:03
Vector都是新買的，每次做都多少會這樣......
推 rebirth: TA vector不加insert直接transform看看？看看有沒有污染10/03 21:10
直接加還是做一個no inserts的ligation controls? 如果測出有問題那這家公司的vecto
r品質堪憂QQ
※ 編輯: ernielwl (175.96.64.191), 10/03/2017 22:11:40
推 rebirth: 或許兩種都該試試，天曉得是DNA fragment還是plasmid 10/03 23:22
→ rebirth: contamination10/03 23:23
→ Sylvaine: 有考慮用其他re切切看嗎？還有，能請教你們用什麼做為t10/04 20:17
其他RE切沒什麼意義，從一開始挑起來的clone裡面insert就錯了
→ Sylvaine: emplate嗎？10/04 20:17
insert是由cDNA為template PCR出來的，內文有說，會經過一次切膠純化，所以片段大小
不會出錯
※ 編輯: ernielwl (175.96.64.191), 10/04/2017 21:55:58
→ blence: 從你放的blast來看,好像是訂序到backbone,也就是pUC19序列10/04 22:40
→ blence: 這1+0.5k不是塞到TA之間,所以應該不是insert污染 10/04 22:41
→ blence: 跑個uncut看有insert的比vector only,或許可以看到雜band 10/04 22:43
→ blence: 只是這是vector或來自Ecoli的污染,要測過才知道 10/04 22:47
→ blence: 我們曾經遇過Ecoli有plasmid污染,就是他們的HIT DH5a10/04 22:51
原來如此，看來他們的 HIT DH5a也可能有問題......
不過我也有用他們這組competent cell 裡面附的 control plasmid，transform出來全部
都是藍色菌落，如果污染到應該也會混白色的
目前推測TA vector裡面污染的可能性比較高

謝謝大家給的建議與回覆!
※ 編輯: ernielwl (175.96.64.191), 10/04/2017 23:46:23






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rbc ta cloning vector 在 【求救】 TA cloning错误insert请教- 生技板 - PTT網頁版 的八卦

请教经验丰富的各位做TA cloning会常常挑到错误的insert吗小弟使用RBC TA cloning kit inserts 大小为1.5k，PCR产物经过gel extraction 纯化 ... ... <看更多>

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