丁香实验_LOGO
登录
提问
我要登录
|免费注册
点赞
收藏
wx-share
分享

琼脂糖凝胶回收DNA――纸浆法

互联网

1301
This procedure isolates DNA from agarose gels by filtration through a filter-paper column. The column is made in a 500 µL tube from a slurry of filter paper in TE buffer.

Materials

1. Whatman 3MM filter paper: 50 cm2 piece
2. TE Buffer (10X): dissolve 186 mg EDTA and 605 mg Tris in 50 mL dH2O; pH to 8.0 with HCl. Store at 4°C, dilute ten times before using.
3. Paper Slurry: cut 50 cm2 of filter paper into tiny (1-2 mm2) pieces; add to 40 mL of TE buffer in a 50 mL tube. Shake vigorously by hand for at least 5 minutes. Store at 4°C.
4. Agarose gel with DNA to be isolated
5. Clean razor blade
6. Filter column: Punch a small hole in the bottom of a 500 µL tube with a 23 gauge needle. Remove paper slurry piecewise with tweezers and place over the hole in the 500 µL tube. Pack with a 200 µL pipet tip and remove excess liquid. Repeat until paper column is approximately 3 mm high. Place inside a 1.5 mL tube to collect eluent.

Procedure

1. Excise the DNA band from the surrounding gel with a clean razor blade; be sure to remove as little gel as possible.
2. Dice the excised gel fragment into small pieces with the same razor; transfer onto filter column.
3. Centrifuge for 10 minutes at highest speed (approximately 20,000g) in a microcentrifuge.
4. Transfer eluent to a fresh tube; recentrifuge agarose
5. Combine eluent with that from previous centrifugation
6. Assemble a second spin column and transfer remaining agarose to the new column. Spin again.
7. Combine all eluent fractions and concentrate via cold ethanol precipitation
提问
扫一扫
丁香实验小程序二维码
实验小助手
丁香实验公众号二维码
扫码领资料
反馈
TOP
打开小程序