tailieunhanh - Molecular Biology Problem Solver 20

Molecular Biology Problem Solver 20. Sách được nhiều nhà khoa học có uy tín, nhiều kinh nghiệm trong nghiên cứu thực nghiệm trình bày những vấn đề thường hay phát sinh trong phòng thí nghiệm. Do vậy mà sách không trình bày các protocol hay quy trình như các sách khác, thay vào đó các tác giả sẽ trình bày các vấn đề nhằm giúp giúp đọc giả: Tự nâng cao khả năng chẩn đoán nguyên nhân khi gặp các vấn để về kỹ thuật, quy trình, hóa chất, thuốc thử trong quá trình thực nghiệm trong phòng thí. | Whenever nucleic acids are denatured there is a risk of irreversible denaturation. Never increase the denaturation time beyond what is recommended and ensure that pH values are accurate for neutralization. Prolonged high pH or heat exposure may lead to more contamination with genomic DNA Liou et al. 1999 and nicked open and irreversibly denatured plasmid. The pH of solution 3 of an alkaline lysis procedure needs to be pH to precipitate out SDS protein genomic DNA. Effects of changing criticial parameters have been studied in detail Kieser 1984 . These protocols have been modified to purify cosmids but larger DNA molecules will not renature as well as small plasmids. Most methods work well for plasmids up to 10kb above 10kb denaturation has to be milder Hogrefe and Friedrich 1984 Azad Coote and Parton 1992 Sinnett Richer and Baccichet 1998 . The yield of low copy number plasmids can be improved dramatically by adding chloramphenicol Norgard Emigholz and Monahan 1979 or spectinomycin 300mg ml Amersham Phar-macia Biotech unpublished observations which prevent replication of chromosomal but not plasmid DNA. However extended exposure to such agents have also been shown to damage DNA in vitro Skolimowski Knight and Edwards 1983 . Resources Plasmid purification methodology could fill an entire book of its own. Traditional chromatography has been applied to isolate large- and small-scale preparations of plasmid from a variety of hosts. Techniques include gel filtration anion exchange hydrophobic interaction chromatography single-strand affinity matrix Pham Chillapagari and Suarez 1996 Yashima et al. 1993a b triple helix resin silica resin and hydroxyapatite in a column as well as microtiter plate format. Plasmid purification procedures are reviewed in O Kennedy et al. 2000 Neudecker and Grimm 2000 Monteiro et al. 1999 Ferreira et al. 2000. Ferreira et al. 1999 Ferreira et al. 1998 Huber 1998 Lyddiatt and O Sullivan 1998 and Levy et al. 2000a . CsCl Purification .