Gel extraction protocol using SPRI

1. Gel Extraction Protocol Using SPRI
1. Weigh gel slice and add 3x volume of NaI Solution. Place sample at 42C shaking
or at 55C water bath for 15mins and invert tube every 3-5mins.
2. Add 5ul of beads + 30-40% IPA and invert tube 10 times, and bind at room
temperature for 5mins.
3. Place tube on SPRI stand for 5mins.
4. Discard SN
5. Wash beads off magnet with 500ul of New Wash solution (Wash buffer). Tipmix
10 times
6. Bind beads on SPRI stand for 1min, and discard SN.
7. Repeat step 5, for a total of two washes.
8. Dry beads on magnet for 5 minutes.
9. Elute with 20-40ul of dH2O, resuspend beads, and sit at room temperature for 3
minutes. Remix sample and bind beads to magnet.
Appendix
NaI Solution:
6M NaI (Qbiogene Geneclean kit)
OR
6M NaI
50mM Tris pH 7
5mM Ascorbic Acid
New Wash (Qbiogene Geneclean kit)
OR
0.15M NaCl
50mM Tris 7.4
10mM EDTA
40% Ethanol
dH2O
Weight of gel slice: 100mg =0.1g
****Magnetic Beads increase by 1ul per 5 to 10ug of starting input. (Have not tested
bead capacity, maximum input tested was 1ug.)
IPA percentage should be calculated per total volume with in the tube.

2. A B C D E F G
A. 5ul beads/New Wash (42%) E. Geneclean protocol (51%)
B. Geneclean protocol (30%) F. 5ul beads/100%IPA wash (62%)
C. 5ul beads/70%ETOH wash (24%) G. 10ul Ampure/70%ETOH wash (65%)
D. 5ul beads/New wash (86%)
40% IPA 30% IPA
Elution volume 22ul, and 5ul of final product on the 4% Egel.
Elution done in 37C water bath
Fluorescence vs. ng
0
20000
40000
60000
80000
100000
120000
140000
0 20 40 60 80 100 120
Series1
65
795.905
36.1775
G
62
1022.98
46.4989
F
51
898.142
40.8246
E
86
800.399
36.3818
D
24
91.7047
4.16839
C
30
126.043
5.72923
B
42
162.419
7.38266
A
%Yield based on DNA
INPUT
total yield
ng/ul
Pico
Green
%Yield Based on purified PCR Product