Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies
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We need to clone two relatively short (1.5 kbp) human genomic DNA fragments. They are from 2 different genes and each contains intron as well as exon sequences. Because of their size, we do not need cosmids or any kind of fancy vector. We have tried to clone them into good-old pBluScript as well as another home-made vector. Nothing has worked. The fragments were amplified by PCR with restriction sites incorporated near the extremities - nothing groundbreaking. We are wondering: 1) does it take a special kind of vector to clone small random human DNA, perhaps because of some sequences present in introns? 2) does it take special E coli strains to grow these plasmids? Thanks for any helpful advice you would have.
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If you can amplify the genomic DNA fragments by PCR, you can clone them in any vector (such as BlueScript) and any E coli host (such as DH5-alpha). The only problem you may face is to cut the fragments with restriction enzymes correctly. I would suggest to clone in EcoRV-cut (or SmaI-cut), dephosphorylated vector via blunt-end ligation. Be sure to use proofreading polymerase (such as Pfu), gel purify the PCR fragment and treat them with T4 polynucleotide kinase (purify with PCR Purification spin column) prior to ligation.
Not affiliated with any company. Mention of a specifc product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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