• Clean electrodes of Pippin Prep by installing cleaning cassette filled with H2O and
closing lid for 30 s. Remove Cassette.
10. Sample Clean-Up
• Use the QiagenQIAquick PCR Purification Kit (cat# 28106) for clean-up procedure.
Perform the PCR Purification Protocol.
• Add 5 volumes of PB buffer (with Indicator) to 1 volume of the Pippin Prep elute
(250μl PB + I buffer +50μl Pippin Prep Elute).
• Check the color of the mixture and ensure that it is yellow (similar to the original
color of buffer ERC).
• If color is orange or violet, add 10μl of 3 M sodium acetate pH 5.0 and mix. The
color will return to yellow.
• Place a MinElute column in a 2 ml collection tube.
• Bind DNA by adding the sample to the MinElute column and centrifuging 1 min at
13,000 RPM at RT˚˜.
• Discard the flow-through and place the MinElute column back in the same
collection tube.
• Wash column by adding 750μl of Buffer PE to the MinElute column and centrifuge
1 min at 13,000 RPM at RT˚˜.
• Discard flow-through and place MinElute column back in the same collection tube.
• Centrifuge MinElute column for an additional 1 min to remove any residual wash
buffer PE.
• Place the MinElute Column in a new labeled 1.5 ml microcentrifuge tube.
• Elute DNA by adding 23μl of EB buffer (10 mM Tris-HCl pH 8.5) to the center of
the membrane. Let column stands at RT˚˜ 1 min.
• Centrifuge MinElute column for 1 min at 13,000 RPM.
• Check volume of eluted samples and verify that 20μl + are obtained. Add resuspension buffer, if needed, to obtain 20μl.
11. Enrich DNA Fragments.
Master mix final PCR (ChIP)
1 x [μl]
x [μl]
PCR Primer Cocktail[7]
5.00
PCR Primer Master Mix[8]
25.00
30.00
206
Appendix
closing lid for 30 s. Remove Cassette.
10. Sample Clean-Up
• Use the QiagenQIAquick PCR Purification Kit (cat# 28106) for clean-up procedure.
Perform the PCR Purification Protocol.
• Add 5 volumes of PB buffer (with Indicator) to 1 volume of the Pippin Prep elute
(250μl PB + I buffer +50μl Pippin Prep Elute).
• Check the color of the mixture and ensure that it is yellow (similar to the original
color of buffer ERC).
• If color is orange or violet, add 10μl of 3 M sodium acetate pH 5.0 and mix. The
color will return to yellow.
• Place a MinElute column in a 2 ml collection tube.
• Bind DNA by adding the sample to the MinElute column and centrifuging 1 min at
13,000 RPM at RT˚˜.
• Discard the flow-through and place the MinElute column back in the same
collection tube.
• Wash column by adding 750μl of Buffer PE to the MinElute column and centrifuge
1 min at 13,000 RPM at RT˚˜.
• Discard flow-through and place MinElute column back in the same collection tube.
• Centrifuge MinElute column for an additional 1 min to remove any residual wash
buffer PE.
• Place the MinElute Column in a new labeled 1.5 ml microcentrifuge tube.
• Elute DNA by adding 23μl of EB buffer (10 mM Tris-HCl pH 8.5) to the center of
the membrane. Let column stands at RT˚˜ 1 min.
• Centrifuge MinElute column for 1 min at 13,000 RPM.
• Check volume of eluted samples and verify that 20μl + are obtained. Add resuspension buffer, if needed, to obtain 20μl.
11. Enrich DNA Fragments.
Master mix final PCR (ChIP)
1 x [μl]
x [μl]
PCR Primer Cocktail[7]
5.00
PCR Primer Master Mix[8]
25.00
30.00
206
Appendix