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Troublshooting failed transformation


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#16 NemomeN

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Posted 27 February 2009 - 04:29 PM

I did a quick check of the pcDM8 plasmid and the plasmid itself does NOT have any selection other than the SupF gene (suppresser tRNA to allow for translation of the mutated AmpR and TetR genes in the p3 episome). The presence of the SupF in pcDM8 allows for translation of mutated AmpR and tetR in the p3 episome (bacterial strain). The p3 episome itself (and subsequently the strain) can be selected using KanR and then AmpR and tetR selection is used only when a SupF plasmid is added.

It may be worth checking to make sure that the plasmid that is being transformed actually contains the SupF gene and has not been altered. What you have been describing sounds like the backbone may no longer contain the SupF marker.....

NK

#17 DNA

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Posted 27 February 2009 - 10:46 PM

I did a quick check of the pcDM8 plasmid and the plasmid itself does NOT have any selection other than the SupF gene (suppresser tRNA to allow for translation of the mutated AmpR and TetR genes in the p3 episome). The presence of the SupF in pcDM8 allows for translation of mutated AmpR and tetR in the p3 episome (bacterial strain). The p3 episome itself (and subsequently the strain) can be selected using KanR and then AmpR and tetR selection is used only when a SupF plasmid is added.

It may be worth checking to make sure that the plasmid that is being transformed actually contains the SupF gene and has not been altered. What you have been describing sounds like the backbone may no longer contain the SupF marker.....

NK



Thanks NemomenN, What do you mean by that the backbone may no longer contain SupF marker, So what happened to the marker, You mean fallen off of plasmid or what? I did not understand. In that case what do i need to do then?

To tell you one more thing, that when i did the control, that also did not work. At the same time i just plated only competent cells with out
antibiotc and there was a lot of growth.

#18 NemomeN

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Posted 02 March 2009 - 09:19 AM

Plating cells without antibiotic will give you colonies since they normally grow. Out of curiosity, what control are you using (is it pcMD8)? If the control is the same backbone, it sounds like the bacteria you are using have lost the p3 episome (or it's no longer working). The p3 episome does have KanR resistance, and you can add some kan to make sure that the p3 is still there. I would try to re-make some competent cells that have been selected with KanR. Outside of that, there's not much more that I can think of other than sublconing your gene into a different vector that has the antibiotic selection on it.

Good luck

#19 DNA

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Posted 07 March 2009 - 09:33 PM

Plating cells without antibiotic will give you colonies since they normally grow. Out of curiosity, what control are you using (is it pcMD8)? If the control is the same backbone, it sounds like the bacteria you are using have lost the p3 episome (or it's no longer working). The p3 episome does have KanR resistance, and you can add some kan to make sure that the p3 is still there. I would try to re-make some competent cells that have been selected with KanR. Outside of that, there's not much more that I can think of other than sublconing your gene into a different vector that has the antibiotic selection on it.

Good luck



Could you give me the different steps involved in subcloning of this gene in to another vector. I wanna use PCDNA3.

Nobody in our lab has done it before so i need some guidance about this process.

Thanks

#20 NemomeN

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Posted 08 March 2009 - 06:46 PM

The easiest way would be to find the sequence of your gene, design primers to amplify it and clone it into pcDNA3 with the appropriate restriction enzymes. Ideally, two diffent ones that do not cut into your gene. This is probably the easiest way in theory to do it unless you have the map of your vector that contains the restriction sites.




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