Troublshooting failed transformation - pLEASE hELP (Feb/19/2009 )

So, the transformation has failed on two different sets of chemically competent cells (one from your lab, and one from another lab)?

What is it you're transfoming with? Is it a ligation mixture? If so, maybe that's your problem...

Try transforming the cells with some known-good uncut vector.

HomeBrew on Feb 22 2009, 05:52 AM said:

No its not a ligation mixture. I am actually transforming Human GABAA alpha2 subunit plasmid cloned in to PcDM8 vector.
Yes i tried the cells of other lab as well but again it did not work. Today i am trying all tubes of my competent cells with controlDNA puc19 ON ampicillin
only plates which are recommended by invitrogen. Second i am trying to plate some untransformed cells from a couple of tubes as well with no antibiotic plates. let see what happens?

Then i would like to order fresh competent ells and will transform with some other plasmids to rule out the question of competent cells.

DNA on Feb 21 2009, 09:34 PM said:

You've cloned a plasmid into another plasmid? How big is the construct? Are the two plasmids compatible?

HomeBrew on Feb 22 2009, 01:36 PM said:

The DNA sequence of GABAA is subcloned in to the vector pcDM8. The size of the plasmid is 4000 kb.

No that i snot a big issue since it has been successfully transformed in teh same condition sin teh past but it is only on this occasion its not working.

I have not done it in teh past, some previuos students have done it.

OK, I did the control with another two tubes and they did grow, but the control is with ampicillin only 25ug/ml.

While my vector confer resistance to both ampicilline and tetracycline, I dont know what to do , should i try transformation with those two tubes of cells with ampicilline only or use both antibiotics?
In case of ampiciline only if i get teh growth, do you think i will get the correct plasmids then?

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

NemomeN on Feb 28 2009, 10:59 AM said:

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.

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

NemomeN on Mar 3 2009, 02:49 AM said:

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

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.