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double digest weirdness

double digest weirdness - Molecular Cloning Forum

double digest weirdness - 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 07-27-2010, 03:13 PM
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Default double digest weirdness



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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