• From Ilana Brito from Alm lab (posted by Sarah Preheim)

Protocol for library whole genome construction

1. Shear DNA by sonication. Make sure your sample is in 50ul of solution. Start with 2-20ug of DNA. Fill BioRupter with water (upto .5 inches from line) and then ice upto line. Do 6 cycles, replace ice. Repeat for a total of 18-20 cycles of 30seconds on/off with “H” setting. Average 200-400 base pairs.

1. 2.     End-repair

• Blunt and 5’-phosporyate the DNA from step 2 using Quick blunting kit.
• Mix:   

sheared DNA (2μg)                 45.5μl

10x Blunting Buffer                6μl

1mM dNTP Mix                    6μl

Blunt enzyme mix                   2.5μl

TOTAL                                  60μl

• Incubate at RT for 30 minutes
• Purify using Qiagen MinElute column (these are kept in the fridge.) Elute in 12μl.
  

1. 3.     Ligate Solexa adaptors

• Solexa adapters must be hybridized before use. Heat to 95 for 5 minutes, cool slowly to Room temperature.
• Ligate adaptors, using a 10x molar excess of each one, and as much DNA as possible.
• Mix:

                                    End-repaired DNA                                         10μl (12.5 pmol)

                                    100μM IGA adapter A#                                 1.25μl (125 pmol)

                                    100μM IGA adapter B#-PE                           1.25μl (125 pmol)

                                    2X Quick Ligation Reaction Buffer (NEB)    15μl

                                    Quick T4 Ligase (NEB)                                  2.5μl

                                    TOTAL                                                          30μl

• Incubate at RT for 15 minutes.

1. 4.     Size selection and purification using SPRI beads.

• Mix DNA and beads to appropriate ratio: 0.65X SPRI beads: Add 19.5 μl of SPRI beads to 30μl reaction from step 3.
• Incubate at RT for 20 minutes.
• Place tubes on magnet for 6 minutes.
• Transfer all SN to new tube. Discard beads.
• Mix DNA and beads to appropriate ratio, 1X SPRI beads: Add 10.5 μl SPRI beads to 49.5μl reaction.
• Vortex, spin.
• Incubate at RT for 7-20 minutes.
• Place tubes on magnet for 6 minutes.
• Remove all SN, keep beads.
• Wash with 500μl 70% EtOH, incubate for 30 seconds, remove all SN.
• Repeat: Wash with 500μl 70% EtOH, incubate for 30 seconds, remove all SN.
• Let dry completely for 15 minutes. Remove from magnet.
• Elute in 30μl EM.
• Vortex.
• Incubate at RT for 2 minutes.
• Put on magnet for 2 minutes
• Transfer SN to new tube.
  

1. 5.     Nick translation

• Bst polymerase can be used for nick translation---it can be used at elevated temperatures which is good for melting and secondary structures and lacks both 3’-5’ and 5’3’ exonuclease activity.
• Mix:

Purified DNA                                                 14 μl

10X Buffer (NEB)                              2μl

10mM dNTPs                                                0.4μl

1mg/ml BSA                                        2μl

Water                                                  0.6μl

Bst polymerase (Enzymatics)                        1μl

TOTAL                                              20μl

• Incubate at 65 degrees, 25 minutes.

1. 6.     Library Enrichment by PCR.

• Perform 2 25μl reactions: (100μM primer)
• Mix:

H2O                                        19.125μl

5X Pfu Turbo buffer               5μl

dNTPs            10mM                         0.5μl

40μl Solexa PCR-A-PE           0.25μl

40μl Solexa PCR-B-PE           0.25μl

SybrGreenI                             0.125μl

Nick-translated DNA             2μl

Pfu Turbo                               0.25μl

TOTAL                                  25μl

• Program:       

1. 95˚C    120sec
2. 95˚C    30sec
3. 60˚C    30sec
4. 72˚C    60sec
5. 95˚C    120sec
6. Go to step 2 34 more times.
7. 72˚C    5 min
8. 4˚C      Forevair

• These 2 reactions are to check cycle time only. Look at the melting curves---use the mid-log point to pick the ultimate cycle time.
• Prep PCR as above, but in 2 100μl reactions using 8μl of sample in each, and cycle with cycle number.
• Mix:

H2O                                        77μl

5X Pfu Turbo buffer               20μl

dNTPs            10mM                         2μl

40μl Solexa PCR-A-PE           1μl

40μl Solexa PCR-B-PE           1μl

Nick-translated DNA             8μl

Pfu Turbo                               1μl

                                                      TOTAL                                  100μl

• Run on a QIAElute column. Elute in 50ul. (You could also do a single SPRI---check the ratios of beads to reaction volume)
• Analyze using Bioanalyzer.