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Help~ Incorrect constructs in cloning - (Nov/01/2013 )



I want to clone a gene (1.5 kb) and amplify it on plasmid by PCR


after PCR, I perform gel extraction cut the PCR product and then digest with SpeI-HF and XhoI about 2 hr


in the some time, I digest my vector with SpeI-HF and XhoI about 2 hr and add CIP 1hr


after digestion, I perform gel extraction both inset and vector


then doing ligation for 5:1=I:V O/N




pick colony and mini-prep


then check plasmid by SpeI and XhoI


BUT result have no postive, most plasmid is not empty vector


your can see Fig

Attached Image


C is postive control


1,2,5,6 seem empty vector


3,4,7,8 ...and very much other colony(not show) is not empty vector and not postive cloning


why and what is that?


thx a lot~~~




You might have ligated the insert in twice. Thus introducing 2 additional restriction sites which would result in 4 bands.


thinks a lot!!


I think so too


but PCR check is negtive


I think if that's 2 fragment insert, PCR will have band~


whatever if that is mean ligated the insert in twice, should I reduce insert in ligation reaction ?


I agree that the PCR should work with the insert ligated twice. But if it does turn out to be a double insertion, just play with the insert:vector ratio. Try a 1:1, or 3:1 vector to insert ratio. If you are not quantifying the insert before ligation (I often dont), it is easy to put way too much insert in the reaction.