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
- 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:
- 98˚C 70sec
- 98˚C 15sec
- 65˚C 20sec
- 72˚C 30sec
- Go to step 2 34 more times.
- 72˚C 5 min
- 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.