DNA浓度测定.ppt

文档介绍：DNA浓度测定
质粒类型：
严谨型：这些质粒的复制是在寄主细胞严格控制之下的，与寄主细胞的复制偶联同步。所以，往往在一个细胞中只有一份或几份拷贝；
松驰型：这些质粒的复制是在寄主细胞的松弛控制之下的，每个细胞中含有10-200份拷贝℃培养12～16小时;
-3ml于Eppendorf管中，10000r/min离心1min，去掉上清液，加入100l溶液1 ，充分混匀;
加入200 NaOH(内含1％SDS)，加盖颠倒6-7次使之混匀，冰上放置5min;
质粒小量提取的方法：
加入150  l乙酸钾溶液()，加盖后颠倒6-7次混匀，冰上放置5－15min;
用台式高速离心机，10000r/min离心7min，上清移入另一干净离心管，并加1ml 100％乙醇混匀，12000r/min离心5min，弃去上清液;
沉淀用70％乙醇清洗一次，小管倒置于吸水纸上，除尽乙醇，室温自然干燥;
加入30-50l灭菌蒸馏水或TE 缓冲液溶解提取物，室温放置15-30min，使DNA充分溶解(有时需要酚:氯仿抽提);
将分离出的质粒DNA置于-20℃保存备用。
WizardTM Plus Minipreps DNAPurification System (Promega)
This purification system is silica membrane-based and modified alkaline lysis procedure based system, which specifically absorbs DNA in the presence of high concentration of some salt. Proteins and other impurities are not absorbed and will be removed. DNA can be eluted under low-salt condition or water.
The yield of plasmid DNA will vary depending on a number of factors.
Cell Resuspension Solution
50mM Tris-HCl (pH )
10mM EDT
100µg/ml RNase A
Cell Lysis Solution
NaOH
1% SDS
Neutralization Solution
potassium acetate (pH )
Column Wash Solution
80mM potassium acetate
Tris-HCl (pH )
40µM EDTA
55% ethanol
TE buffer (1X)
10mM Tris-HCl (pH )
1mM EDTA
注意：
用移液器（俗称“枪”）操作时，每一步都要格外小心，以免切碎DNA(包括质粒DNA和基因组DNA);
加入溶液II 和III后，一定不要剧烈振荡，避免导致基因组DNA的断裂而污染质粒DNA！！！
Pellet 1–3ml of cells by centrifugation for 1–2 minutes at 10,000 × g in a microcentrifuge. Pour off the supernatant and blot the tube upside-down on a paper towel to remove excess media.
Completely resuspend the cell pellet in 200µl of Cell Resuspension Solution (Solution I）.
Add 200µl of Cell Lysis Solution (Solution II) and mix by inverting the tube 4 times. The cell suspension should clear immediately.
Add 200µl of Neutralization Solution (Solution III)and mix by inverting the tube 4 times.
Centrifuge the lysate at 10,000 × g in a microcentrifuge for