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deleting lacZ gene from control vector


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5 replies to this topic

#1 SF_HK

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Posted 10 February 2009 - 02:02 AM

Hi,

I have deleted the lacZ gene from the pcDNA3.1(-)/myc-His/LacZ vector to be able to clone my gene it it. Has anyone tried this.I'm actually out of pcDNA3.1()-/myc-His/ vector and hence I'm using the control vector be deleting teh reperter gene. Has anyone does this? ANy precautios to be taken? My insert is 1052bp long.

#2 scolix

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Posted 10 February 2009 - 05:10 AM

yeh, its quite normal for people to substitute the original gene for another one. Just take care of the orientation of the new gene.

#3 cellcounter

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Posted 10 February 2009 - 06:28 AM

yeh, its quite normal for people to substitute the original gene for another one. Just take care of the orientation of the new gene.

And that it should be in frame with the tags.

#4 SF_HK

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Posted 10 February 2009 - 06:54 AM

yeh, its quite normal for people to substitute the original gene for another one. Just take care of the orientation of the new gene.

And that it should be in frame with the tags.


Thank you very much for your response. My only concern was that I was making use of the control vector provided by Invitrogen since I ran out of another vector. I was not sure if there would be any problem in deleting a reporter gene (e.g. LacZ ) from control vector and replacing it with my gene on interest.

control plasmid offered by Invitrogen: pcDNA3.1(-)/myc-His/LacZ i.e. 8.8kb
plasmid I have ran out of: pcDNA3.1(-)/myc-His/ i.e. 5.8kb

As you can see, the lacZ gene is quite big. I have removed this from teh control vector and replaced it with 1052bp of insert.

#5 perneseblue

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Posted 10 February 2009 - 08:51 AM

might you still have the "empty" tube which you kept the pcDNA3.1(-)/myc-His/ plasmid?

There is usually enough plasmid DNA on the walls of that tube too successfully transform the plasmid back into cells. Just mix the competent cell with the empty tube and transform.
May your PCR products be long, your protocols short and your boss on holiday

#6 Nghia

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Posted 04 June 2009 - 03:20 PM

Dear All,

Please help... I need to delete LacZ from a following construct: -----Enhancer--Promoter---LacZ---SV40-----

How do I delete the entire LacZ gene without affecting of construct? If anyone has such kind of any experience on this,
would you please give me some suggestions? That will be very helpful.

Thanks.




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