quick cloning question - (Aug/13/2005 )
I'm about to perform my first real cloning experiment and I have a quick question. I'm double-digesting my vector so that sticky ends complementary to my insert of interest are generated. However, am I supposed to purify the larger vector fragment from the smaller one following the digestion? Otherwise it would seem like the unwanted insert would compete with the insert I'm attempting to subclone.
You can avoid vector reconstitution by adding shrimp alkaline phosphatase (SAP) or calf intestinal alkaline phosphatase (CIP) to remove 5' phosphates on your digested vector. You can then use polynucleotide kinase (PNK) and ATP to phosphorylate your insert of interest. With an insert that has 5' phosphates and a linear vector that lacks 5' phosphates, a bridge can form betwee the two and can be sealed up using T4 DNA ligase.
Hope this helps!
Use gel electrophoresis to separate the vector from the unwanted fragment, then do a gel extraction using a kit like qiagen make.
You may not even need to phosphorylate/dephosphorylate. I'd try it w/o those steps first so as to save some supplies.