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家喻戶曉新蟲 (小有名氣)
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[求助]
菌檢 已有1人參與
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我之前用一對引物58℃擴啟動子,也擴出來了,現(xiàn)在連T載,挑陽性克隆,為什么一直沒有,是不是加有酶切位點的引物的退火溫度應(yīng)該提高一點。 發(fā)自小木蟲Android客戶端 |
木蟲 (正式寫手)

新蟲 (初入文壇)
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???????taq????????м?a?????????????{??Y????????? insert??3 ?|?w??1??λ??mole ????С???Android????? |
新蟲 (小有名氣)
新蟲 (小有名氣)
新蟲 (小有名氣)
新蟲 (初入文壇)
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???f?????u?????????e???x????????{??Y??????|?????????????????x???????colony PCR ?????????LB plate?? PCR????insert????L?????????? ??_??????????????????|?w ????С???Android????? |
新蟲 (初入文壇)
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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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