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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.


Digested plasmid

Digested plasmid - Molecular Cloning Forum

Digested plasmid - 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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Old 07-21-2013, 11:27 AM
Pipette Filler
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Default Digested plasmid



I have a plasmid with size of 2744bp (PENTR11) and containing an insert (429bp). I have did double digestion (Not1 and Noc1) so that I can put my promoter next(upstream) to my insert. When I ran the gel to check my digested plasmid, I got 1 single band with size around 5kb for sample A. Meanwhile for sample B, I got same size of band as sample B with kb and an addition 2 bands with size around 2kb and 3kb.These additional bands are very thin and almost invisible. Both sample A and B are same, just duplicate. I'm not sure if I have successful digested my plasmid or not. And 1 more thing, I didn't put any loading buffer, I straight away put 1uL of my digested plasmid and ran the gel.

I used 14uL of plasmid, 4uL of NEB buffer 3, 1uL of Noc1,and 1uL of Not1. Then incubate at least 3 hours at 37oC. Then I add 2uL of antartic phosphatase buffer and 1Ul of antartic phosphatase and incubate at 37oC for another 30min. Then inactivated at 65oC for 10min.
I have attached my gel and with hyperladder 1
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File Type: jpg RE 1D & 3B 210713.jpg (8.9 KB, 2 views)
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