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Separation of Primers from Vector

Separation of Primers from Vector - Molecular Cloning Forum

Separation of Primers from Vector - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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  #1  
Old 02-22-2011, 01:14 PM
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Default Separation of Primers from Vector



In a mixture of complimentary primer and vector, will agarose gel electrophoresis separate them if they have annealed but not ligated? Also in a transformation of unligated vector can bacteria take up primer annealed but not ligated to a complimentary vector?

My reason for asking is I have primers designed for regular mutagenesis which I am now using with a different system. These primers are overlapping and produce sticky ended vectors, for various reasons this system you are supposed to produce blunt ended. However I'm on a very tight budget and would like to save myself the expense of reordering these primers if possible.
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Old 02-22-2011, 03:37 PM
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Default Re: Separation of Primers from Vector

Hi Helen welcome!

When you mean annealed but not ligated do you mean no ligase (ie no enzymatic ligase action occurred) was added?
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Old 02-23-2011, 03:39 PM
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Default Re: Separation of Primers from Vector

Yes- so they are stuck together by complimentary base pairs but there is no actual covalent bond between them.




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Old 02-23-2011, 11:49 PM
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Default Re: Separation of Primers from Vector

Hi Helen,
I believe so as nicked vector also runs on an agarose gel on its own band as well. Also I have annealed primers together to make them double stranded and did not use ligase to keep them together

The only thing I do not know is if you can separate annealed DNA (I guess it depends on the size difference) versus non-annealed to see if there is a difference or to confirm. IE I have no idea at the moment if a single stranded primer will run different than a double stranded primer, but at least on gels only double stranded primers show up (primer dimer). The issue is how to confirm you have annealed samples (or even if you need to)?

I would try with a small sample, then try more if everything works ok.

Last edited by admin; 02-23-2011 at 11:51 PM.
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Old 02-24-2011, 11:12 AM
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Default Re: Separation of Primers from Vector

The problem with the system I am using is it requires the primers to be 5'-phosphorylated so they are annealing to the overhang on the vector and getting ligated in causing duplicate regions. I need to get rid of them- either by skipping the ligation step and transforming directly or by gel electrophoresis and extraction. But will they separate if annealed.

These are normal mutagenesis primers. They reccomend designing ones with no overlap to make a blunt ended vector- presumably to avoid this problem. However I am on a stretched budget and would like to avoid ordering new ones if possible.
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Old 02-25-2011, 10:38 AM
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Default Re: Separation of Primers from Vector

I guess your question is will the primers separate from each other if annealed - no - they shouldn't separate from each other. I used to anneal my primers by making sure they were single stranded, then getting them under ideal conditions to anneal. Also make sure you designed them well - I doubt they will not anneal unless they are at high temperature conditions.

if your question is will they separate out on a gel, I answered that a bit above - I think they should separate from non-annealed primers no problem on a gel
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Old 03-01-2011, 04:18 PM
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Default Re: Separation of Primers from Vector

Transforming directly without a ligation step got rid of the duplicated region although there were a lot few colonies.

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Old 03-02-2011, 05:06 AM
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Default Re: Separation of Primers from Vector

great to hear Helen,
good luck with the sequencing!

let us know
best wishes
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