Materials Needed:

-       Bio-Rupter (Parson’s 3rd floor in between Polz and Chisholm labs)

-       Quick blunting and ligation kit (NEB, E0542L 100RXNs $473.60)

-       10mM dNTP mix ( NEB, N0447L $216.00)

  • also need a 1:10 dilution for 1mM dNTPs

-       IGA adapter A# (10uM working solution)

-       IGA adapter B#-PE (10uM working solution)

-       SPRI beads (Beckman Coulter, A63882, 450mL $4,100)

-       BST Polymerase large fragment (NEB, M0275S 1,600U $49.60)

-       IGA-PCR-PE-F primer (40uM working solution)

-       IGA-PCR-PE-R primer (40uM working solution)

-       Phusion, with HF buffer (NEB, M0530L 500U $329.60 )

-       SybrGreen (Invitrogen S7563 500uL 10,000x $235.00 )

-       Qiaquick PCR cleanup column (Qiagen, 50 columns: 28104, $98.94; 250 columns: 28106, $465.60)

-       MinElute Reaction clean up column (Qiagen 50 columns: 28204, $109.61; 250 columns: 28206, $503.43)

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. Use Agilent Bioanalyzer to confirm shear size.

 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. 

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 

                                    100μM IGA adapter A#                                 1.25μl

                                    100μM IGA adapter B#-PE                           1.25μl 

                                    2X Quick Ligation Reaction Buffer (NEB)       15μl

                                    Quick T4 Ligase (NEB)                                 2.5μl

                                    TOTAL                                                          30μl

  • Incubate at RT for 15 minutes.

 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. 

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.

 6.     Library Enrichment by PCR.

  • Perform 2 25μl reactions:
  • Mix:

H2O                                        16.6μl

5X HF Buffer                           5μl

dNTPs (10mM)                        0.5μl

40μM Solexa PCR-A-PE           0.25μl

40μM Solexa PCR-B-PE           0.25μl

SybrGreenI                             0.125μl

Nick-translated DNA                2μl

Phusion                                0.25μl

TOTAL                                  25μl

  • Program:       
  1. 98˚C    70sec
  2. 98˚C    15sec
  3. 65˚C    20sec
  4. 72˚C    30sec
  5. Go to step 2 34 more times.
  6. 72˚C    5 min
  7. 4˚C      Forever
  • 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                                        66.8μl

5X HF Buffer                           20μl

dNTPs(10mM)                         2μl

40μM Solexa PCR-A-PE           1μl

40μM Solexa PCR-B-PE           1μl

Nick-translated DNA                 8μl

Phusion                                   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.