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transformation problem


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8 replies to this topic

#1 iamleos

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Posted 29 May 2013 - 11:16 PM

i am trying to clone my gene in pTZ. when ever i do transformation in DH5 alpha strain i get nothing on transformed plates. am doing transformation by T/A cloning method. my ligation is ok. my cells are fine. but still cant get results. can any one guide me?

#2 Missle

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Posted 30 May 2013 - 05:08 AM

What is your transformation procedure?

#3 iamleos

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Posted 30 May 2013 - 07:24 AM

i am trying to clone my gene in pTZ. when ever i do transformation in DH5 alpha strain i get nothing on transformed plates. am doing transformation by T/A cloning method. my ligation is ok. my cells are fine. but still cant get results. can any one guide me?

#4 Missle

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Posted 30 May 2013 - 09:04 AM

What is your transformation procedure? There are lots of potential problem areas and knowing your basic procedure will help.

#5 DrLeo

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Posted 30 May 2013 - 07:51 PM

1. You should check all the material: X-gal, Agar plates, SOC medium... if their quality is good or not. It's easy to use the positive control where you can use the transformed CP cells with known plasmid DNA.
2. How do you know your ligation was good?
3. You should mention your protocol so that other members can figure out what problem is.

#6 iamleos

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Posted 30 May 2013 - 08:52 PM

i am using instaclone pcr cloning kit. first i make competent cells in C-medium of the kit. then make their pellet and add T solutions as written in protocol. with addition of ligation in cells and T soln. i spread them on agar plates containing IPTG, X-gal and ampicillin. incubate them over night at 37 C. result is always no growth. i have checked my protocol. gave my ligation and cells to one of my friend she easily transformed it. our procedure is same.
i am not getting where i am doing wrong.

#7 iamleos

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Posted 30 May 2013 - 08:54 PM

i am using instaclone pcr cloning kit. first i make competent cells in C-medium of the kit. then make their pellet and add T solutions as written in protocol. with addition of ligation in cells and T soln. i spread them on agar plates containing IPTG, X-gal and ampicillin. incubate them over night at 37 C. result is always no growth. i have checked my protocol. gave my ligation and cells to one of my friend she easily transformed it. our procedure is same.
i am not getting where i am doing wrong.

#8 notjustalabtech

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Posted 09 June 2013 - 11:29 AM

are you adding enough cells to your plate? Try plating everything rather than just 100 ul of your transformation. spin it down to reduce volume first.

#9 DrLeo

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Posted 09 June 2013 - 11:23 PM

Did You culture your CP cells with or without plasmid??? If your CP cells did not contain any plasmid with Amp-resistant gene, they can not grow on Amp(+) agar...




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