Chelex-100法提取微量DNA

Chelex-100法提取微量DNA

1. Add 1/50 volume of Proteinase K (10 mg/mL) and incubate the sample at 65°C for 2 hrs to overnight.

2. 200 uL 5% Chexlex-100,add 10 uL proteinase K (20 mg/mL),inculate 2 hrs in 37 °C.

3. 50ul Chelex液及lfl蛋白酶K水，56％温水浴消化2h后，煮沸8min，迅速放人冰盒冷却4min，以5000r／min速度离心lmin。吸取上清液作为DNA扩增模板于～20％保存备用。

4.All bacterial strains were treated in 400 μl of 5% Chelex 100 buffer (containi ng 0.03% SDS, 1% Tween-20 and 1% NP40) and boiled for 15 min. DNA of each sam ple was amplified by removal of some supernatant. In order to lyse Staphylococc us, proteinase K was added to the 5% Chelex 100 buffer, and the mixture was incu bated at 56℃ for 60 min and then boiled for 15 min. Blood samples in the prese nce of heparin, were subjected to red cell lysis in five volumes of 0.87% NH?4 Cl, leukocytes were pelleted, suspended in 200 μl of 5% Chelex 100 buffer with 20 mg/ml proteinase K, incubated at 56℃ for 60 min and then boiled for 15 min, centrifuged for 1 min, at which point the supernatant was collected for PCR amp lification. CSF samples were centrifuged for 5 min. Then the supernatant was p oured off and the cell pellets were resuspended in a mixture, and treated in the same method as the blood samples.

5.提取毛发等物的DNA（刑事鉴定）

（1）取一小块冰冻的组织（少于1mg，可用经消毒的枪头钻取），或1～3 根带有毛根的毛发（需先经 90％、70％的乙醇和灭菌水依次清洗，并剪去毛干部分），或 1～5µl 血液，加入经过高温消毒的离心管中。离心管内事先加入 500µl 5％的Chelex 溶液。

（2）将样品管在56℃下放置1 小时或更长时间，直至组织样品成粉末状（不同组织所需时间各不相同）。

（3）在振荡器上振荡10～15 秒钟。

（4）在 95～100℃下煮 15～40 分钟。

（5）在振荡器上振荡10～15 秒钟。

（6）将样品管在4℃下保存，进行PCR 反应之前再次离心，使Chelex 颗粒沉淀。取上清液（1～10µl）作为DNA 模板。

根据以上资料设计的盘菌DNA提取方法：

1. 8~10个子囊盘,加7~10粒石英砂,置液氮中冷冻5~10分钟,取出在冰上研磨

2. 加200 ul 5% Chelex,震荡10 s.

3. 37℃温浴过夜.

4. 震荡10 s.

5. 99.9℃ PCR 12 min,取出10000 rpm ,离心10 min .

6. 取5 ul 上清液作模板DNA,PCR扩增.

7. 1.5%回收胶,150伏10 min .

8. 用百泰克回收试剂盒进行回收.