7 replies to this topic

[Khushboo Agrawal]

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.

[grvsomani]

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.

[Curtis]

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plasmid extraction you get no plasmid at all, meaning they were false colonies I discovered that when my ampicilin is out of date this happens a lot.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.

[Khushboo Agrawal]

grvsomani, on 24 May 2011 - 06:02 AM, said:

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.

[Khushboo Agrawal]

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.

[Khushboo Agrawal]

Curtis, on 24 May 2011 - 07:38 AM, said:

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.

[Khushboo Agrawal]

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.

[phage434]

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.