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cloning 1052bp fragment - no colonies (Feb/10/2009 )

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SF_HK on Feb 20 2009, 04:53 PM said:

That is an interesing way. But won't the ligation reaction give a smear when running on agarose gel? One silly question, you woulb be adding the loading dye to the ligation reaction before loading to the gel?

No, it looked like a plasmid band, which is higher than the vector and I DID use the loading dye ^^. Anyway, consider it as the plasmid and everything will be clear


SF_HK on Feb 17 2009, 04:56 AM said:

I did a control reaction by adding ligase and single digested plasmid incubated at 16 deg for 16 hours. Followed by transformation. this did not give me any colonies.

Does hat mean that the ligase is not working.

I used the same liagese and liagated double digetsed plasmid(50ng) with my insert (200ng), 4deg 16hours, this gave 5 colonies but all empty?! So, what were those colonies if they didn't even have the self ligated vector?

I guess there is some problem with the ligase or the single digest of the plasmid has overdigested the ends. This can also prevent ligation.

We typically ligate at RT for 30min. and for normal ligations start with 20ng of vector and 1:3 or 1:5 ratio of inserts.

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