A noncommercial polymerase chain reaction-based method to approach one hundred percent recombinant clone selection efficiency.

Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.

Routine cholecystectomy is not mandatory during morbid obesity surgery.

BACKGROUND: Controversy exists concerning the utility of routine cholecystectomy during bariatric surgery. We report our series of bariatric surgical procedures at our institution without concurrent cholecystectomy. METHODS: From October 2003 to August 2005, 621 morbidly obese patients underwent a weight loss operation. Preoperatively, each patient had undergone abdominal ultrasound (AUS) to evaluate for abnormal gallbladder findings. Patients with previous cholecystectomy were excluded. Symptomatic patients with AUS findings consistent with gallbladder disease underwent concomitant cholecystectomy and bariatric surgery. Asymptomatic patients, despite AUS findings, did not undergo cholecystectomy with their bariatric operation. A comparison between the preoperative AUS-positive and AUS-negative, asymptomatic patients after bariatric surgery was performed. RESULTS: Of the 621 patients who underwent bariatric surgery, 170 (27%) had undergone previous cholecystectomy and were excluded. Of the remaining 451 patients, 17 with positive AUS findings and symptoms underwent cholecystectomy during bariatric surgery. The range of follow-up was 4-25 months. Of the 451 patients, 324 were asymptomatic and had negative AUS findings and 102 were asymptomatic and had positive AUS findings for gallbladder abnormalities. Postoperatively, 29 asymptomatic/AUS-negative patients (9%) developed symptoms and had positive AUS findings. Nine asymptomatic patients with AUS positive findings (9%) developed symptoms. Finally, 38 patients (8.4%) went on to undergo elective cholecystectomy. These 2 groups were not signficantly different statistically. CONCLUSIONS: In this study, the development of symptomatic/AUS-positive gallbladder abnormalities was low after obesity surgery, suggesting that mandatory cholecystectomy is not required at bariatric surgery.