Genomic DNA Sample Preparation

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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-5P
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Genomic DNA Sample Preparation
3. Add A Bases to the 3 End of the DNA
Fragments
P5-AGTCTTGGATCGAC-3 3-TCAGAACCTAGCTG-5P
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-5P
4. Ligate Adapters to the DNA Fragments
P5-AGTCTTGGATCGACA-3 3-ATCAGAACCTAGCTG-5P
Adapters are ligated to the ends of the DNA
fragments, preparing them to be Hybridized to the
flow cell
P5-AGTCTTGGATCGACA-3 3-ATCAGAACCTAGCTG-5P
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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 150-200bp, while 300-650bp is a long
insert template
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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-5P
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-5P
Extension
P5-AGTCTTGGATCGACA-3
3-NNNNNTCAGAACCTAGCTGTNN-5
5-NNTAGTCTTGGATCGACNNNNN-3
3-ATCAGAACCTAGCTG-5P
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Genomic DNA Sample Preparation
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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 500-1000ng of DNA.
• Measure the 260/280 ratio. It should be 1.8-2.0.
• 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