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Can you show me your agarose gel eletrophoresis of PCR products? Which polymerase did you use? Was there one A added at 3'-termini?
3Â¥2015-12-30 20:34:46
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4Â¥2015-12-30 20:54:29
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3Â¥: Originally posted by ·´Ó¦Á´ at 2015-12-30 20:34:46
Can you show me your agarose gel eletrophoresis of PCR products? Which polymerase did you use? Was there one A added at 3'-termini?

I used GXL ploymerase .One A was added at 3¡®-termini usingTaq
ploymerase.
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5Â¥2015-12-30 21:30:23
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4?: Originally posted by ayumi7667 at 2015-12-30 20:54:29
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6Â¥2015-12-30 21:32:37
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2Â¥: Originally posted by ¿ÆÑ§Ð¡ñû at 2015-12-30 20:11:08
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7Â¥2015-12-30 21:35:35
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???f?????u?????????e???x????????{??Y??????|?????????????????x???????colony PCR ?????????LB plate?? PCR????insert????L?????????? ??_??????????????????|?w

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8Â¥2015-12-30 22:02:01
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8?: Originally posted by ayumi7667 at 2015-12-30 22:02:01
???f?????u?????????e???x????????{??Y??????|?????????????????x???????colony PCR ?????????LB plate?? PCR????insert????L?????????? ??_??????????????????|?w
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9Â¥2015-12-30 22:03:31
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6Â¥: Originally posted by ¼ÒÓ÷»§Ïþ at 2015-12-30 21:32:37
ÎÒÊÇÓÃTaq¼ÓA£¬Ã»ÓÐÓÃÀ¶°×°ßÌô¿Ë¡£¬µ«ÊÇÎÒËæÒâÌôÑ¡¼¸¸ö°ßÒ¡¾úÌáÖÊÁ£²¢²âÐòµÄ»°£¬½á¹ûÒ²ÊÇÁ¬Éϵ쬵«ÊǾú¼ì¾ÍÊÇûÓУ¬¶øÇÒ²âÐò½á¹û¶¼Óмî»ùÍ»±ä...

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
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