June, 1997 by Hans

Prepare a homemade spin filter (column) for DNA purification

Materials
o
§ GF/F glass fiber membrane filter (furnace at 500 C)
§ 2.0, 1.8 and 0.5ml of microcentrifuge tubes
§ 1000ul blue tip
§ Needle
§ forceps
§ bunsen burner

Procedures
1. Cut blue tip by three parts as picture below and the middle part of tip is not needed.

2. put the end part into the top part as picture below and insert this into the 1.8ml of
centrifuge tube. This will be used for crushing agarose gel. Put pieces of gel into the blue
tip and centrifuge it. Agarose gel will be crushed easily. If you use QG buffer(Qiagen),
you don't need to proceed this step.

3. torch the needle for making holes.

4. Make 3-4 holes at the bottom of 0.5ml tube.

5. if you worry about the contamination, burn GF/F glass filter in the furnace at 500 degree
C.
6. Fill the GF/F filter into 0.5ml tube shown picture as below.
 7. 2.0ml tube can be used as well.

Note: if you press the filter too firm, you will need a long time for spin. Just use 5mm diameter of
filter for making column.

Gel extraction kit (DNA fragment recovery from agarose gel)

Buffer QG (neutralization and binding buffer)
5.5M guanidine thiocyanate (GuSCN)
20mM Tris-HCl pH 6.6 (25℃)

Buffer PE (wash buffer)

20mM NaCl
2mM Tris-HCl pH 7.5 (25℃)
80% ethanol

Buffer EB (elution buffer)

10mM Tris-HCl pH 8.5 (25℃)
PCR purification kit
Buffer PB (DNA binding buffer)
5.5 M guanidine hydrochloride (GuHCl)
20mM Tris-HCl pH 6.6 (25℃)

Buffer PE (wash buffer)

20mM NaCl
2mM Tris-HCl pH 7.5 (25℃)
80% ethanol

Buffer EB (elution buffer)

10mM Tris-HCl pH 8.5 (25℃)
Plasmid extraction kit
Buffer P1 (cell suspension buffer)
50mM Tris-HCl pH 8.0 (25℃)
10mM EDTA
50ug/ml of RNase A
Buffer P2 (lysis buffer)
0.2M NaCl and 1% SDS

Buffer N3 (neutralization and binding buffer)

4M guanidine hydrochloride (GuHCl)
0.5M potassium acetate pH 4.2

Buffer PB (wash buffer)

5M guanidine hydrochloride (GuHCl)
20mM Tris-HCl pH 6.6 (25℃)
38% ethanol

Buffer PE (wash buffer)

20mM NaCl
2mM Tris-HCl pH 7.5 (25℃)
80% ethanol

Buffer EB (elution buffer)

10mM Tris-HCl pH 8.5 (25℃)