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Help~ Incorrect constructs in cloning


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#1 uuffoo2222

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Posted 01 November 2013 - 04:28 AM

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

圖片1.jpg

 

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~~~

 

 



#2 HOYAJM

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Posted 01 November 2013 - 09:36 AM

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



#3 uuffoo2222

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Posted 02 November 2013 - 07:23 AM

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 ?



#4 HOYAJM

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Posted 04 November 2013 - 08:45 AM

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.






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