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Removing sites from binary Vector - Help!

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#1 izzybusydizzy



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Posted 22 February 2011 - 02:46 PM

Good day everyone, I can use your help.

I have a binary vector that contains two restriction sites close together that I need to remove so that they can be used later in cloning.

The sites are AsiSI GCG AT /CGC and SwaI ATTT / AAAT

I took my vector and cut it with SwaI overnight, followed by AsiSI overnight. I proceded to Fill in the AsiSI site with Klenow.
I then gel purified my fragment (~8kb) and used 50ng to ligate it to itself, using NEB's Quick ligase kit (50ng of vector 10min room temperature).

I tranformed 2ul of the reaction into Top 10 Cells and I got 0 colonies. I also transformed my cut vector with no ligase and got 0 colonies. I decided to check if my ligation worked by running it on a gel.

I ran the rest of my ligation on a gel along with the original vector (not cut), my cut vector/AsiSi/SwaI/Klenow and the reaction I ligated.

Lane1 = Uncut vector
Lane2 = vector/AsiSi/SwaI/Klenow
Lane3 = Ligation reaction

On the gel I see that my ligation reaction shows a band much larger than the vector/AsiSi/SwaI/Klenow reaction!!!
The ligation reactions shows no band at the size of the vector/AsiSi/SwaI/Klenow nor a smaller band at the original vector (no cut).

I am not sure what this could mean? I could have expected no ligation at all = band at size of vector/AsiSi/SwaI/Klenow
I could have expected ligation to work = band around the size of the original (uncut) vector

I don't know what this band means. It is 2X+ the size of the vector/AsiSi/SwaI/Klenow.

Any ideas? of what could have happened?

Is my plan correct? But with desired enzymes - blunt with Klenow - gel purify - ligate - voila!!! vector minus those sites?
Am i missing something?

Please help, thank you!


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