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Molecular Cloning Forum 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.


Need a cloning genius

Need a cloning genius - Molecular Cloning Forum

Need a cloning genius - 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 03-20-2014, 01:29 PM
Pipette Filler
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Default Need a cloning genius



Hi, I have a 600bp PCR product that I need to attach 55base 5' single-stranded DNA overhangs to (both strands). Any ideas how best to do this? Thankyou
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Old 03-21-2014, 01:57 AM
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Default Re: Need a cloning genius

55 bases of poly T or A or any combination of ATCG?
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Old 03-21-2014, 02:25 PM
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Default Re: Need a cloning genius

No, the overhangs need to be of a particular sequence.

To put into context, I have used Cas9 nickase activity to cleave the genome where I want to create a knock in. The nature of my Cas9 digest has created two 5' overhangs of 55bp. I need to clone 55bp overhangs complementary to the genomic overhang to aid ligation-gated recombination by non-homologous end joining. The sequence cannot be altered as it is within the open reading frame of the gene, and I need it to be expressed.
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Old 04-01-2014, 04:01 AM
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Default Re: Need a cloning genius

Sorry for the late reply.

Sadly at the moment, the answer is beyond me.
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