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Reasons for failure of transformation


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11 replies to this topic

#1 Khushboo Agrawal

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Posted 24 May 2011 - 05:00 AM

I had tried to transform plasmid DNA carrying gene resistance to kanamycin and observed many visible colonies also on antibiotic selected plate after 24 hours of incubation but I am always unsuccessful to isolate the correct plasmid DNA which I transformed into competent DH5a cells instead there is lot of genomic DNA from bacterias on isolation. I had interpreted from my results that colonies on transformation plates are not correct.I also prepared mini preps of DNA by innoculating several single colonies in LB broth but could never get the correct plasmid when I checked my results after restriction digestion on agarose gel. I am unable to find the reason for not getting the correct plasmid DNA. Is it possible that my competent DH5a cells are not working properly? But if this is the reason than why I am able to see the positive colonies on antibiotic plate, are the colonies on plate are just of E.coli cells which are not actually transformed because as i think its not possible for normal E.coli cells to grow in prescence of kanamycin. If anyone can suggest me the reason of my failure it will be a great help.

Edited by Khushboo Agrawal, 24 May 2011 - 05:10 AM.


#2 grvsomani

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Posted 24 May 2011 - 06:02 AM

actually many time chemical competent e.coli cells donot work well, they do not take particular plasmid. same problem i have also faced when i was regularly using stbl3 cells but recently i have failed more than 5 times to clone my insert than i used xl-10 gold ecoli cell that worked well.
another thing i want to ask u that are u getting specific specific plasmid band in gel after isolation. if u r using conventional miniprep method (alkaline lysis)then try it with kit/column purification . otherwise do rnase treatment after plasmid isolation.

#3 Curtis

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Posted 24 May 2011 - 07:38 AM

this is a common problem. Many people face it. some times you get colonies, but after plasmid extraction you get no plasmid at all, meaning they were false colonies I discovered that when my ampicilin is out of date this happens a lot. So I change my ampicilin stock every 1-2 months. I have never had this problem with Kanamycin though. it is one of my favorite antibiotics.

Edited by Curtis, 24 May 2011 - 08:07 AM.


#4 Khushboo Agrawal

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Posted 24 May 2011 - 11:57 PM

actually many time chemical competent e.coli cells donot work well, they do not take particular plasmid. same problem i have also faced when i was regularly using stbl3 cells but recently i have failed more than 5 times to clone my insert than i used xl-10 gold ecoli cell that worked well.
another thing i want to ask u that are u getting specific specific plasmid band in gel after isolation. if u r using conventional miniprep method (alkaline lysis)then try it with kit/column purification . otherwise do rnase treatment after plasmid isolation.



#5 Khushboo Agrawal

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Posted 25 May 2011 - 12:14 AM

Thanks really so much for your help. I want to tell you that after using alkaline lysis for preparing DNA mini prep I finally grow them in large volume of LB medium and isolate using invitrogen mega-kit which already contains RNAse so I think the problem is not with this step. The other thing I want to tell you is that I had recently prepared my DH5a competent cells and I think they have good efficiency, also in datasheet of my plasmid its suggested to use DH5a cells. Yesterday I performed transformation again using a commersialized plasmid to be sure if problem is with my methodology or the plasmid. I prepared positive(kanamycin) and negative(ampicillin) control both and in morning and I observed high number of colonies on plate with kanamycin as my plasmid carries genes resistance to kanamycin but absolutely no growth on ampicillin plate. So can I be sure that my competent cells are working properly and there is some other problem. If not please tell me the ways to check the competency of my cells.

#6 Khushboo Agrawal

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Posted 25 May 2011 - 12:15 AM

this is a common problem. Many people face it. some times you get colonies, but after plasmid extraction you get no plasmid at all, meaning they were false colonies I discovered that when my ampicilin is out of date this happens a lot. So I change my ampicilin stock every 1-2 months. I have never had this problem with Kanamycin though. it is one of my favorite antibiotics.



#7 Khushboo Agrawal

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Posted 25 May 2011 - 12:18 AM

Thanks for your help. I want to tell you that I have freshly prepared my ampicillin and kanamycin stock. Also my plasmid carries genes resistance to kanamycin so there is no role of ampicillin in case of my plasmid. If you have any other suggestions please tell me. Also If you can tell me the ways to check the competency of my bacterial cells it would be a great help.

#8 phage434

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Posted 26 May 2011 - 12:18 AM

Very likely your competent cells are not sufficiently competent. People think that if they can transform prepared plasmid with their competent cells that they are good enough for ligation. This is not true. You need to test your cells for competence. The way to do this is to serially dilute a test plasmid to 10 pg or 100 pg/ul. Transform with 1 ul of this dilute solution and count transformants. Express as transformants/ug of DNA used. You should get 10^8 - 10^9 cfu/ug. Most "competent cells" prepared by novices get 10^6/ug and rarely work for transforming ligation products.

#9 Shrubal

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Posted 13 March 2014 - 02:22 AM

Hi.

 

Was a solution to this found? I seem to be having the same problem - "transformation" and growth on kanamycin plates (freshly prepared; 100ug/mL) and broth containing kanamycin. But when I do a mini-prep, and perform restriction digestion (double digest with two different enzymes), I don't get a band of the right size as compared to the non-recombinant plasmid. I actually see digestion, but the band is way lower than the one seen in my non-recombinant lane. I've repeated this thrice (from glycerol stocks, streaked out cultures and broth cultures of the original "transformants") and I keep getting the same result.

 

advice please?



#10 phage434

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Posted 13 March 2014 - 10:31 AM

We have far too little information to help you. Tell us the details of what plasmids, how the insert is prepared, what enzymes, how you are ligating, what primers are used if any, basically everything you can think of. It sounds on the surface as if it could be that you are ligating primer-dimers, but we need a LOT more information.



#11 Shrubal

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Posted 15 March 2014 - 04:34 AM

I'm sorry I didn't give more information:

 

Vector: pET28a(+)

Insert: PCR amplicon (I included the restriction sites plus 5 more nucleotides on either side of the primers)

Restriction enzymes: NcoI and XhoI

Cloning host: E.coli DH5alpha

Antibiotic selection: 100ug/mL Kanamycin

 

I double digest the vector and insert overnight at 37oC, run an agarose gel and elute the digests from it. I then carry out ligations with the gel eluates overnight at 16oC. When I transformed the competent cells (They're good preps because I always run an uncut vector transformation as a control), I got about 10 clones which I then transferred from plates containing Kanamycin to broth containing the same amount of Kanamycin. There was good growth overnight at 37oC and so I proceeded to isolate the plasmids from them. That's where the problem began -  I get a good plasmid prep but when I digest the plasmids, I can see that there is digestion but the bands are way lower than where they should be (almost at the size of the insert). I can't be transforming insert dimers because then the cells would simply not grow in the presence of Kanamycin.

 

The only difference I see when I isolate the plasmids from the "recombinant" and non-recombinants is that I always see the nicked plasmid band in the non-recombinant, but never in the "recombinant". Always.

 

It's clear therefore that I am missing something but I can't figure out what. I've checked that both my restriction enzymes and my ligase (and buffers) are working with lambda DNA.



#12 phage434

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Posted 15 March 2014 - 06:43 AM

I suspect that the plasmids you are recovering from the Kan plates are either the original uncut plasmid, or (more likely) ligation of a PCR primer-dimer into the cloning site. In both cases, you will see small fragments when the plasmid is cut, and no insert. Is the gel for your pcr product showing a clean band with little or no short fragments showing? If not, then I suggest you concentrate on improving your pcr reaction (change annealing temperature, redesign primers). You might find the primer analysis tools at IDTDNA.com useful. Particularly look at the heterodimer results.

 

Are you doing a control where you ligate and transform without the insert present? This would be useful in determining what is happening.

You can't tell anything about the quality of your competent cells by transforming uncut plasmid, unless it is diluted to 10-100 pg/ul level, and transformed with 1 ul.






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