Docstoc

Genomic DNA Sample Preparation

Document Sample
Genomic DNA Sample Preparation Powered By Docstoc
					                     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’
  Convert the overhangs from fragmentation into         3’-ACCTAGCTG-5’
  Blunt ends, using T4 polymerase & E. coli DNA
  Polymerase I Klenow fragment

  The 3’ to 5’ exonuclease activity of
  these enzymes removes 3’ overhangs
                                                  P5’-AGTCTTGGATCGAC-3’
  & the polymerase activity fills in the 5’        3’-TCAGAACCTAGCTG-5’P
  overhangs
                    Genomic DNA Sample Preparation


3. Add ‘A’ Bases to the 3’                           P5’-AGTCTTGGATCGAC-3’
   End of the DNA Fragments                           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       P5’-AGTCTTGGATCGACA-3’
 have a single ‘T’ base overhang at their 3’ end     3’-ATCAGAACCTAGCTG-5’P



4. Ligate Adapters to the DNA                      P5’-AGTCTTGGATCGACA-3’
   Fragments                                       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
                                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
                                   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        P5’-AGTCTTGGATCGACA-3’
ends, & to amplify the amount of DNA in the          3’-ATCAGAACCTAGCTG-5’P
library. The PCR is performed with two primers
that anneal to the ends of the adapters                               Denaturation


                                                     P5’-AGTCTTGGATCGACA-3’
Amplify using the following PCR protocol:                 Primer Annealing
*30 seconds at 980C                                  3’-ATCAGAACCTAGCTG-5’P
*18 cycles of:
  10 seconds at 980C
  30 seconds at 650C                                                     Extension
  30 seconds at 720C
*5 minutes at 720C
                                                       P5’-AGTCTTGGATCGACA-3’
*Hold at 40C
                                                 3’-NNNNNTCAGAACCTAGCTGTNN-5’
                                                   5’-NNTAGTCTTGGATCGACNNNNN-3’
                                                      3’-ATCAGAACCTAGCTG-5’P
                        Genomic DNA Sample Preparation
                                                                       1     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 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

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:2
posted:3/6/2012
language:
pages:5