The kan-sacB cassette was liberated from pKSF-II [19], [20] by sequential double digestion with XhoI (New England Biolabs) and SmaI (New England Biolabs), and this fragment was ligated to the appropriately digested pDSM386 to create pDSM387.
The PCR products were fused together by sequential overlap extension, digested with XhoI and NotI, and ligated into pKS149 cut with SalI and NotI to give pKS152.
The two PCR products were fused together by sequential overlap extension, digested with MfeI and HindIII (pyc, aceE, and pgi fragments) or with HindIII and EcoRI (ppc and ilvE fragments), and ligated into pK19mobsacB cut with EcoRI and HindIII.
Protein extracts from subclones of iGFP-WT and iGFP-NLSm cell lines were obtained by sequential extraction with RIPA followed by urea buffer and analyzed by immunoblot.
Briefly, subchondral bone specimens were digested by sequential collagenase type I digestion, followed by cell culture in BGJb medium containing 20% FBS.
Gastric digests were adjusted to pH 6.0 with sodium phosphate buffer and subjected to a simulated duodenal digestion by sequential addition of the pancreatic enzymes trypsin (0.375 mg/mL) and chymotrypsin (0.375 mg/mL) at 37°C for 30 min (both from Sigma) (17).
Neurons from either cortex or thalamus were purified from P1 hevin KO pups by sequential immunopanning as follows: Following dissection, cortex/thalamus was digested for 30 min in papain (Worthington, NJ).
Isolated fractions were dried by vacuum centrifugation, digested with trypsin, and fractionated by reverse phase HPLC.
Chondrocytes were isolated by sequential pronase (70 U ml−1, 1 hour at 37°C) and collagenase (300 U ml−1, 3 hours at 37°C) digest.
