5 replies to this topic

[SF_HK]

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.

[scolix]

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

[cellcounter]

scolix, on Feb 10 2009, 06:10 AM, said:

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.

[SF_HK]

cellcounter, on Feb 10 2009, 10:28 PM, said:

scolix, on Feb 10 2009, 06:10 AM, said:

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.

[perneseblue]

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.

[Nghia]

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.