No colony came out on the selective plate - (Nov/02/2004 )

Hello everyone. I want to ligate a 2kb fragment with pSE129 (12kB) plasmid. The plasmid were double-digested with Xba1 and Kpn1 (Both restriction enzyme are Invitrogen products). Fragment with cohesive end for Xba1 and Kpn1 was PCR-amplified and TA cloned, then degested with both Xba1 and Kpn1. After I was sure that plasmid and fragment were successively digested, I performed ligation overnight using invitrogen T4 ligase @ room temperature, control was intact plasmid. Unexpectedly, no colony came out on the plate for plasmid+fragment ligation, however, so many colonies grew on the control plate. I am at a complete loss. What happened to me.

I am needing your powerful help!!!! the clock is running fast!!!

Narrowriver

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Hi Narrowriver,

Did you purify your restricted insert and plasmid before ligation? Is your ligase and its buffer are still good? How about run a gel after ligation to see if the ligation is successful (use reaction without ligase as control).

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Hi, pcrman, thank you so much for your message. Actually I gel-purified digested plasmid and insert before ligation. 100ng plasmid (about 12kb) and 100ng insert (2kb) were used for ligation reaction. Invitrogen T4 ligase and its buffer are brand new. I appreciate your good idea on running a gel for ligation verification.

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