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Troublshooting failed transformation


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19 replies to this topic

#1 DNA

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Posted 19 February 2009 - 09:18 PM

hI aLL GEEKS,

I have done transformation using MC1061/P3 ultracomp E.COLI with tetracyline (10ug/ml) ampicillin (25ug/ml).
I did teh positive control as well. I used diffirent dilutions of DNA, diffrent medium like NZYME AND SOC, but none of them worked.
Everything was used fresh, like plates, antibiotics and medium etc. New tube of competent cells was opened.

Can anyone suggest something what could be teh possible reason?

Do you think my competent cells are no longer competent though they have been purchased just last year in august from invitrogen and since then kept them in -80C FREEZER.

#2 pcrman

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Posted 19 February 2009 - 09:45 PM

If you did not get any thing from your positive control, you have to check whether your competent cells are OK, and your transformation was properly done (right temperature...) and your plates have the right concentration of antibiotics, your incubator is at the right temp.

by the way, what plasmid is pLEASE :lol: ?

#3 DNA

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Posted 20 February 2009 - 04:58 PM

If you did not get any thing from your positive control, you have to check whether your competent cells are OK, and your transformation was properly done (right temperature...) and your plates have the right concentration of antibiotics, your incubator is at the right temp.

by the way, what plasmid is pLEASE :wacko: ?



To check teh competency of competent cells, i transformed with competent cells from some other department too and again that failed. Every thing was right i guess, 30 minutes incubation, heat shcok for 40 seconds at 42C , medias, receommended antibiotic concentration.

The plasmids is human GABAA alpha2 subunit cloned in to pcDM8 vector.

#4 pcrboy

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Posted 20 February 2009 - 11:51 PM

try transforming without antibiotics, maybe your concentration of antibiotics is lethal

#5 DNA

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Posted 21 February 2009 - 12:05 AM

try transforming without antibiotics, maybe your concentration of antibiotics is lethal



Do you think that will give me my desired plasmids? Won,t there be a chance of contamination?

#6 HomeBrew

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Posted 21 February 2009 - 12:30 AM

Did you allow a growth period (say 30 minutes at 37C with shaking) after transformation but before plating? This is required with tetracycline due to phenotypic lag, or your transformants will die. You might also consider dropping the tetracycline to 5 ug/ml; some resistance genes don't convey resistance to 10 ug/ml. Why is your ampicillin concentration so low? I usually select for ampicillin resistance with 100 ug/ml...

#7 DNA

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Posted 21 February 2009 - 12:56 AM

Did you allow a growth period (say 30 minutes at 37C with shaking) after transformation but before plating? This is required with tetracycline due to phenotypic lag, or your transformants will die. You might also consider dropping the tetracycline to 5 ug/ml; some resistance genes don't convey resistance to 10 ug/ml. Why is your ampicillin concentration so low? I usually select for ampicillin resistance with 100 ug/ml...




Yes, i allowed 1 hour incubation at 37 Co before plating.
I have used the antibiotics concentration recommended by invitrogen for MC1061/P3 chemically competent cells.
I cant see any growth at all on the plates.

I have tried using the glycerol stock as well for growth in LB medium using tehsame antiviotic concentration but no growth.

#8 pcrboy

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Posted 21 February 2009 - 01:08 AM

looks like everything is coming out negative, try something that is considered a positive control. which is why i suggested growing without antibiotics.

competent cells should stay fine at -80, so that isnt my major concern. reason is you're assuming your bacteria has recovered from competency while at -80...which suggest they have undergone a lot of growth. try growing untransformed competent cells and transformed cells in no antibiotic media. let's see whats positive and whats negative.

#9 DNA

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Posted 21 February 2009 - 01:12 AM

looks like everything is coming out negative, try something that is considered a positive control. which is why i suggested growing without antibiotics.

competent cells should stay fine at -80, so that isnt my major concern. reason is you're assuming your bacteria has recovered from competency while at -80...which suggest they have undergone a lot of growth. try growing untransformed competent cells and transformed cells in no antibiotic media. let's see whats positive and whats negative.



Thanks PCRBOY for your kind suggestion. I will try that without antibiotics too but i have tried the positive control using ampicilline only plates according to teh recommendations of the invitrogen but that did not work too.

Now i think teh only option is to grow them wd no antibiotic plates.

#10 pcrboy

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Posted 21 February 2009 - 01:18 AM

the media you grow them in should also have no antibiotics. also, i wouldnt screen bacteria that has been grown in normal media, reason is because as you mentioned earlier, there is high risk of contamination.

#11 HomeBrew

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Posted 21 February 2009 - 12:22 PM

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.

#12 DNA

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Posted 21 February 2009 - 06:34 PM

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.



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.

#13 HomeBrew

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Posted 21 February 2009 - 07:06 PM

I am actually transforming Human GABAA alpha2 subunit plasmid cloned in to PcDM8 vector.


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

#14 DNA

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Posted 21 February 2009 - 08:11 PM

I am actually transforming Human GABAA alpha2 subunit plasmid cloned in to PcDM8 vector.


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



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.

#15 DNA

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Posted 22 February 2009 - 04:21 PM

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?




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