I have tried to transform several different plasmids into chemically competent DH5 alpha cells (commercial and hand made), however none of the plasmids transform in to the DH5alpha cells.
My positive control, pUC19, transformed into the cells with a efficiency of 1.6X10^7. However, my ligated sample (using T4 ligase), a 10X dilution of the ligated sample, and uncut plasmids will not transform into the cells. All of the plasmids are ampicillin resistant and I have transformed the vector backbone (plasmid) and a plasmid containing the insert into E.coli before and the transformation has worked (i.e. the backbone and insert are not toxic). I was given three plasmids (i.e. not prepared by me) and miniprepped the rest of the plasmid from already transformed cells so I am not convinced sample prep is the problem.
Any ideas of why pUC 19 (and my lab mates plasmids) will transform, but not my DNA?
Hi Jessica,
I can't give you any definitive answers but here's some thoughts.
10^7 is an OK efficiency, however for ligations I would be happier to see 10^8 or better as there can be large variations in how well the ligation has worked and the quality of the ligated DNA.
It is possible that there is more than one problem here.
The DNA ligation could be not working or be poor, so that doesn't produce any colonies. If you can try using the best efficiency commercial cells (better than 10^8). Ligating more concentrated DNA might also help.
Supercoiled DNA will tranform E.coli much better than the other forms. Have you checked the quality of the uncut plasmids on a gel? Also does the quantity you see on the gel match what you would expect to see from their concentrations?
-Richard
Just a thought. Do your plasmids contain a ccdB gene (a la Gateway destination vectors, for instance)? If so, they should not grow in DH5 alpha.
Richard,
I ran the ligation reaction on a gel and have confirmed that the ligation was successful. Also, I have tried to transform 5 different, undigested plasmids and none of them yielded colonies.I have confirmed the quality and quantity of the plasmids on a gel. The only success I have had with transforming the DNA was with pUC 19.
Alejandro,
I have already transformed the vector and a vector that contains the insert into the DH5 alpha and the cells grew fine.
I am repeating every step in and then giving the DNA to another lab group to transform for me.
Thanks for your input!
Hi Jessica,
that does sound strange. Some further thoughts:
Are the other plasmids also high copy number like pUC19? I'm wondering if they need a lower concentration of antibiotic.
Do you let the cells recover for a good hour at 37oC with shaking before plating them out? If this is shorter then they might not have repaired and started expressing the resistance.
Given that you are using several plasmids that are known to work, I don't think gene toxicity is your problem, but it could be if you still have problems with the ligation cloning after sorting out the transformation.
If you could provide exact details of what you have done, perhaps there's something we can spot.
-Richard
Hi jessica
I hope you are successful in this stage..
I recommend you
1.Check all of your kit( we problem in this stage and wondering)
2.Use formula for ligation
3.Notice that DH5 a grow in 16 h in 37 C ( it is better)
4.Check T4 ligase
5.Try when you cut gel containing your DNA, then use kit for isolation DNA, you must complete dissolve your gel,(if gel big for micro tips 2 ml, use bigger micro tips)
And for increasing concentration your DNA grow minimum 25 ml DH5 a and shed all of them on column
Regards
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