Tris饱和苯酚(Tris saturated phenol)也称Tris平衡酚(Tris balanced phenol),是添加了抗氧化剂8-羟基喹啉,tris pH8.0充分平衡的苯酚,呈黄色。
问: 对比了DNA ladder的实验方法和一些公司的dna ladder提取试剂盒,发现关键的区别就在回收DNA LADDER的柱子上。由于经费有限,请问提取质粒或是回收试剂盒中的柱子能够用于回收DNA LADDER吗?
答:
实验操作步骤:
1、 Harvest cells and pre-cold PBS rinsing once, 500 g centrifuge 5 min Room Temprature
2、 Pellet resuspended in 250 μl TE buffer containing 1μg/μl RNase A
3、 Cells were lysed by adding an equal volume of 1.2% SDS and gentle mixing by inversion(6-8 times)
4、 Incubate 5 min, 350 μl precipitation solution were added, mixed 8-10 times and put on ice for 15 min
5、 Spin 14000g for 15 min Room Temperature and clear supernatant (700 μl) transfer to miniprep column
6、 Spin at 14000 g for 1 min, washed with 700 μl wash buffer once
7、 Spin empty column to discard the rest ethonal
8、 30-50 μl TE(pH8.0) elute the DNA fragments
9、 1.6% agarose gel separated at 30V for 10min, and 80 V for 50 min
10、 EB staining and photographed
实验材料与试剂配制:
1、 TE buffer
2、 CsCl containing precipitation buffer:3M CsCl,1M Potassium acetate,0.67M acetic acid
3、 Wash buffer:80mM potassium acetate,10mM Tris-Cl,pH7.5,40uM EDTA,60% ethanol
4、 1.6% Agarose gel
5、 Miniprep column (QIAprep Spin Miniprep Kit, QIAGEN)
6、 ethidium bromide (10 μl/100ml)
我们实验室是这样做的,如果药物或者处理能够诱导凋亡,都还做得蛮好的,供参考。