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家喻户晓

新虫 (小有名气)

[求助] 菌检 已有1人参与

我之前用一对引物58℃扩启动子,也扩出来了,现在连T载,挑阳性克隆,为什么一直没有,是不是加有酶切位点的引物的退火温度应该提高一点啊!

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新虫 (正式写手)

引用回帖:
6楼: Originally posted by 家喻户晓 at 2015-12-30 21:32:37
我是用Taq加A,没有用蓝白斑挑克隆,但是我随意挑选几个斑摇菌提质粒并测序的话,结果也是连上的,但是菌检就是没有,而且测序结果都有碱基突变...

Was it your first time of screening for recombinants? You did get the resulting recombinants afer TA-cloning, though the colony PCR was false negative. Therefore, something wrong might happen while you made colony preparation for colony PCR.

A well isolated colony was picked and transferred to 50ul of sterile water, boiled for 10 minutes, centrifuged at 12000rpm for 5minutes, and 2ul of the supernatant was used in each amplification (20ul volumes in total).

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  • 2015-12-30 22:55:47, 415.77 K
10楼2015-12-30 22:56:04
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科学小覃

木虫 (正式写手)

【答案】应助回帖

感谢参与,应助指数 +1
T载体连接出现假阳性是常有的事,PCR只要有条带大小正确基本没问题,主要是纯化回收过程中,DNA一定要纯,连接时片段至少是载体的3倍以上。
生物科学
2楼2015-12-30 20:11:08
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新虫 (正式写手)

Can you show me your agarose gel eletrophoresis of PCR products? Which polymerase did you use? Was there one A added at 3'-termini?
3楼2015-12-30 20:34:46
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ayumi7667

新虫 (初入文坛)

???????taq????????м?a?????????????{??Y????????? insert??3 ?|?w??1??λ??mole

????С???Android?????
4楼2015-12-30 20:54:29
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