1. Genomic DNA Sample Preparation
1.Nebulisation of genomic DNA
Double stranded genomic DNA is
broken into random sized fragments of
between 100 to 1000bp
2. Perform End Repair
5’-AGTCTTGGATCGACTCT-3’
3’-ACCTAGCTG-5’
Convert the overhangs from fragmentation into
Blunt ends, using T4 polymerase & E. coli DNA
Polymerase I Klenow fragment
The 3’ to 5’ exonuclease activity of
these enzymes removes 3’ overhangs
& the polymerase activity fills in the 5’
overhangs
P5’-AGTCTTGGATCGAC-3’
3’-TCAGAACCTAGCTG-5’P

2. Genomic DNA Sample Preparation
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
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

3. Genomic DNA Sample Preparation
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
You can select more than one size-range of
Adapter-ligated DNA by excising slices from different parts of the gel. A short insert template is bp, while bp is a long insert template

4. Genomic 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’
Primer Annealing
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
3’-ATCAGAACCTAGCTG-5’P
Extension
P5’-AGTCTTGGATCGACA-3’
3’-NNNNNTCAGAACCTAGCTGTNN-5’
5’-NNTAGTCTTGGATCGACNNNNN-3’
3’-ATCAGAACCTAGCTG-5’P

5. Genomic DNA Sample Preparation1 2 3
7. Validate the Library
Perform the following quality control steps on the DNA
Library:
Determine the library concentration by measuring its
absorbence at 260nm. The yield from the protocol
should be between ng of DNA.
Measure the 260/280 ratio. It should be ~
Either load 10% of the volume of the library on a gel
and check that the size range is as expected, or run the
DNA library on an Agilent 2100 Bioanalyzer.
Determine the molar concentration of the library ready for
Cluster Generation
1: Low molecular weight ladder
2: Long insert DNA library
3: Short insert DNA library