趙明明磁珠純化的原理PPT學習教案

1、會計學1膠回收膠回收玻璃奶法玻璃奶法玻璃奶的制作1.石英砂磨成325目的玻璃粉（研磨2小時左右）;粉末顆粒的大小，指的是粉末可以通過325目的篩網(wǎng)2. 50克玻璃粉懸于加有200mL蒸餾水的燒杯中,攪拌混勻后,靜置90min;3.將上清轉(zhuǎn)移至離心管中6000g離心10min;4.倒掉上清,將玻璃粉重懸于1.5mL 25%的硝酸中,室溫放置過夜; （新購置的玻璃器皿含有較多的游離堿，需要在酸中浸泡數(shù)小時，浸泡后再沖洗干凈）5. 6000g離心10min,沉淀玻璃粉;6.倒掉上清,將玻璃粉用無菌雙蒸水洗滌4-6次,直至pH值中性;7. 用無菌雙蒸水重懸玻璃粉,配成50%勻漿;8.等分成100l樣品

2、, 貯存于-70;待用的樣品可貯存于-20或4.第1頁/共12頁切取目的DNA條帶溶膠結(jié)合玻璃奶離心沉淀玻璃奶洗脫DNA漂洗玻璃奶操作步驟操作步驟第2頁/共12頁原理原理玻璃奶(Glassmilk) 是一種白色硅顆粒，能結(jié)合至50kb大小的DNA（雙鏈、單鏈、線性、環(huán)狀或超螺旋）。 DNA與玻璃奶結(jié)合原理 在高鹽狀態(tài)下，玻璃奶周圍的負電荷被打破，并允許DNA的磷酸負電荷與玻璃奶特異性結(jié)合。在低鹽（H2O或1TE）時溶解分離。鹽濃度 DNA100bp:低鹽狀態(tài)下結(jié)合率高。pH值 :結(jié)合率高。第3頁/共12頁第4頁/共12頁第5頁/共12頁樣品起始樣品起始100l，去除大片段后去除大片段后155

3、l，largetargetWhat plays the role in size selection is not AMPure beads, but its buffer.第6頁/共12頁stock buffer: 20% PEG 8000, 2.5M NaCl, Tris第7頁/共12頁Roche第8頁/共12頁12 l product was mixed with 12 l of 12p XP beads and incubated at RT for 6 minutes. The supernatant was then mixed with 12 l of AMPure XP bea

4、ds and 5 l of 40% of PEG8000 and eluted with 10 l of nuclease-free water, it was then again purified using 12 l of AMPure XP beads and eluted in 30 mL of EB buffer.For size-selection using the SPRI beads, we replaced the stock buffer of AMPure XP beads with a 12% PEG-8000 and 2.5 M NaCl solution (12p XP buffer). One mL of Ampure XP beads were placed on a magnetic stand and left for 10 minutes. Thesupernatant was then removed and the beads were washed twice with ultra-pure