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

hello~everyone

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

transform

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

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.