1. ChIP DNA Sample Preparation
Customers provide us with
ChIP enriched, qPCR verified
DNA, approximately 10ng in
30ul of water.
Zymo Research (
can supply a ChIP DNA clean &
concentrator kit.

2. ChIP DNA Sample Preparation
2. Perform End Repair
5’-AGTCTTGGATCGACTCT-3’
3’-ACCTAGCTG-5’
Convert the overhangs from into phosphorylated
blunt ends, using T4 polymerase & E. coli DNA
Polymerase I large fragment (Klenow polymerase), &
T4 polynucleotide kinase (PNK).
P5’-AGTCTTGGATCGAC-3’
3’-TCAGAACCTAGCTG-5’P
The 3’ to 5’ exonuclease activity of
these enzymes removes 3’ overhangs
& the polymerase activity fills in the 5’
Overhangs.
3. Add ‘A’ Bases to the 3’
End of the DNA Fragments
P5’-AGTCTTGGATCGAC-3’
3’-TCAGAACCTAGCTG-5’P
An ‘A’ base is added to the 3’ end of the
blunt phosphorylated DNA fragments, using
the polymerase activity of Klenow fragment
(3’ to 5’ exo minus). This prepares the DNA
fragments for ligation to the adapters, which
have a single ‘T’ base overhang at their 3’ end
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P

3. ChIP DNA Sample Preparation
4. Ligate Adapters to the DNA
Fragments
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
Adapters are ligated to the ends of the
DNA fragments, preparing them to be
hybridized to the flow cell
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
5. Purify Ligation Products
The products from the ligation are purified on a
2% agarose gel, to remove all unligated adapters,
Remove any adapters that may have ligated to one
Another, & select a size range of templates to go on
The cluster generation platform
Excise a band in the gel from the 200 +/- 25bp range. Also excise a gel slice from an
empty well in the same size range, to be used as a negative control.

4. ChIP DNA Sample Preparation
6. Enrich the Adapter-Modified DNA Fragments by PCR
PCR is used to selectively enrich those DNA
fragments that have adapter molecules on both
ends, & to amplify the amount of DNA in the
library. The PCR is performed with two primers
that anneal to the ends of the adapters
P5’-AGTCTTGGATCGACA-3’
3’-ATCAGAACCTAGCTG-5’P
Denaturation
P5’-AGTCTTGGATCGACA-3’
Amplify using the following PCR protocol:
*30 seconds at 980C
*18 cycles of:
10 seconds at 980C
30 seconds at 650C
30 seconds at 720C
*5 minutes at 720C
*Hold at 40C
Primer Annealing
3’-ATCAGAACCTAGCTG-5’P
Extension
P5’-AGTCTTGGATCGACA-3’
3’-NNNNNTCAGAACCTAGCTGTNN-5’
As a negative control, perform PCR
amplification on the sample extracted
from the empty well.
5’-NNTAGTCTTGGATCGACNNNNN-3’
3’-ATCAGAACCTAGCTG-5’P

5. ChIP DNA Sample Preparation
7. Validate the Library
The amount of starting material is very low (10ng), and after 18 cycles of PCR the yield could still be
too low to see on a regular gel, even though it is enough for cluster generation. The more sensitive
method of quality control analysis is to run the library on an Agilent 2100 Bioanalyzer, to check the
size, purity and concentration. Can not use an OD260/280 ratio for concentration measurements,
since this will not distinguish dsDNA from primers, and therefore cannot be used to validate the
library.
High MW
Marker
Fluorescent Units (FU)
Sample
Low MW
Marker
Fragment Size (bp)
Ladder
Sample