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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. |
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| Hey....I've been having issues with a double digest/insertion and wanted some feedback. Here goes: Enzymes: PinAI and XhoI (both NEB) Used NEB buffer "B" from table After PCR, insert was PCR purified with Invitrogen PCR Gel kit After digestion w/enzymes, insert and vector were cleaned with Invitrogen PCR clean-up kit Plates and growth: 1. bugs only: no growth 2. bugs + vector without phosphatase treatment: lots of growth; lawn 3. bugs + vector w/phosphatase treatment + insert: intermediate growth 4. bugs + vector + insert w/phopahatase treatment: intermediate growth (about same as #3). We have been unable to get a vector with insert after mini and test cut. Phosphatase treatment is new, but I don't get why there was (1) still a lot of growth and (2) ANY growth with insert treated Last edited by palindrometric; 07-27-2010 at 03:15 PM. |
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| digest , double , weirdness |
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