Step	Problem	Possible Reason	Solution
9	Big fragments >700 bp or intact genomic DNA after sonication	Lysis was not efficient enough	Make sure to lyse the sample on ice for 10 min.
		Inefficient sonication	Increase the sonication time in 25 second increments.
9	Fragment size are too small (All less than 200 bp)	The samples are over sonicated	Reduce the sonication time in 25 second increments.
48	Step1&2 did not amplify anything after 25 cycles	1st step priming reaction did not work	Make sure that right primer is used and that diluted Sequenase enzyme is used in the 2nd, 3rd and 4th steps. Increase the Primer 1 concentration Make sure that the thermal cycler lid is set to “Off”
		2nd step amplification reaction did not work	Increase the Primer 2 concentration
48	Amplified DNA after Step1&2 runs higher than expected	Samples are under sonicated.	Phusion polymerase is highly processive and tends to better amplify longer fragments. Increase the sonication timing to ensure shorter fragmentation.
		Fragments are self annealed in the second step	Increase the annealing temperatures in the second amplification step. Use 55 and 60 °C instead of 45 and 50 degrees. Alternatively, use only one annealing temperature of 55 °C.
50	Amplified ChIP DNA is not enriched	The antibody is not specific	Make sure that the antibody is a validated antibody in a conventional ChIP experiment from large number of cells.
		Too much background	Increase the washing stringency. After addition of each washing buffer, incubate for 5 min at 4 °C on a rotating platform.
64	Low DNA yield in the Libraries	Inefficient BciVI digestion	Start the digestion with new fresh enzyme
		High adapter concentration in the ligation step	Decrease the adapter concentration 5 fold (50 fold dilution instead of recommended 10 fold)
		PCR reaction did not work	Double check the reagents and use new PCR mix.
64	Libraries are not enriched by QPCR	ChIP enrichment failed	Assess ChIP enrichment after Step 2 with short amplicon primers by QPCR to make sure ChIP experiment is working.
		Unspecific DNA amplification	Run ~80 ng of the library DNA on agarose gel and make sure that it contains a smear DNA fragments ranging from 250–700 bp but not a single band