Linearization of construct

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vkn111
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Linearization of construct

Post by vkn111 » Tue Feb 19, 2008 8:11 pm

Hi
I have a problem with the linearization of plasmid construct for transfection. After digesting the plasmid construct with RE, the expected fragments were 12.5 kb and 2 kb. I do the gel extraction to purify the 12.5 kb fragment. After purifying the 12.5 kb fragment by Qiagen gel extraction kit, when I checked the DNA by analytical gel, I could see the fragment of exactly 12.5 kb. But, when I precipitate this DNA with 3M sodium acetate, pH 5.2 and Ethanol, a second fragment of 4 kb appears on analytical gel. I was wondering why and how this second fragment appears. It would be nice if anyone could help me with this.

Cat
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Post by Cat » Tue Feb 19, 2008 9:38 pm

Most likely either sodium acetate or ethanol is contaminated. Try to do precipitation on pure water and see if you see 4 kb band.

vkn111
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Post by vkn111 » Sat Feb 23, 2008 5:47 am

Thanks Cat. I will try this.

vkn111
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Re: Linearization of construct

Post by vkn111 » Mon Feb 25, 2008 9:37 pm

Hi Cat,
I tried this and I could solve this problem. Thanks for your suggestion.

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