Kun:LabNotes/ExonomeSeq/2010-1-22
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Test the N2 adaptor protocol for library construction[edit]
- In this protocol, captured PCR amplicons are digested with Mme I. The digested DNA fragments carry a 2bp overhang at the 3'-ends, and a phosphate group at the 5'-end. Such fragments can be ligated with a Y-shape adaptor with a degenerate 2bp overhang at the 5'-end, amplified with the standard Illumina PCR primers, and sequenced.
- In this experiment, I'll test this protocol with one exome library (CV-iB-AB from Alice) and one BSPP library.
1st round PCR[edit]
- I need to amplify enough DNA for Mme I digestion.
x 2 Template 1ul 2X iProof MasterMix 100ul 100uM AmpF6.3 1ul 100uM AmpR6.3 1ul 50X SYBG I 1.6ul 98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10 sec) x 8 -> 72C 2min
- Purified with Qiaquick columns.
- Measured with Nanodrop:
- CV-iPS-B: 116ng/ul x 30ul
- BSPP-B2: 112ng/ul x 30ul
Mme I digestion[edit]
- Calculation of Mme I:
- One unit of Mme I can digest 1ug of phiX 174 DNA.
- phiX 174 DNA contain 5 Mme I cutting sites in 5389bp.
- One captured PCR amplicon contain 2 Mme I cutting sites in ~250bp.
- To cut 1ug of the PCR amplicon, we need 2/250/(5/5389)=8.6 units of Mme I.
DNA 10ul 10X NEBuffer 4 4ul Mme I 4ul 1mM SAM 3ul (I use 75uM since the tube of SAM has been used before) H2O 29ul 37C 1h -> Purified with Qiaquick columns
- Measure concentration with Nanodrop:
- CV-iPS-B: 23ng/ul x 30ul
- BSPP-B2: 26ng/ul x 30ul
- Measure concentration with Nanodrop:
File:2010-01-22-MmeI-digestion.png
Ligation[edit]
- Prepare the adaptor:
100uM PE_N2_adaptor 20ul 100uM PE_b_A 20ul 10x Stoffel buffer 10ul H2O 50ul 94C 2min -> 0.2C/sec to 20C -> 4C hold.
- Set up the ligation reaction. Assuming one ligation site per 100bp dsDNA, adaptor:target=20:1
Digested DNA (~200ng) 10ul 2X QuickLigase buffer 25ul 20uM annealed adaptors 6ul QuickLigase 2.5ul H2O 7ul RT for 15min -> Purified with the MinElute columns, eluted with 15ul EB.
PCR[edit]
x 2 Template 10ul 2x iProof mix 100ul Solexa_PCR_up (10uM) 0.8ul Solexa_PCR_lo (10uM) 0.8ul H2O 89ul 50X SYBG I 0.8ul 98C 30sec -> 8 cycles of (98C 10sec -> 62C 20sec -> 70C 15 sec) -> 72C 2min Add additional cycles: 4 cycles of (98C 10sec -> 70C 15 sec) -> 72C 2min File:2010-01-26-ligation-PCR.png File:2010-01-26-ligation-PCR-real-time-curves.png
Size selection and quantification[edit]
File:2010-01-27-size-selection.png File:2010-01-28-library-quant.png Gel quantification: Library conc(ng/ul) conc(nM) KZ-Exome-CViB-N2-Jan28 5.2ng/ul 31nM KZ-BSPP-B2-N2-Jan28 2.1ng/ul 10nM KZ-DigiQ-FS-300p-Jan27 0.6ng/ul 2.8nM KZ-DigiQ-FS-600p-Jan27 1.3ng/ul 6.0nM
Conclusion[edit]
- Library construction seems to be successful. There are clean smears at the expected size range.
- Ideally, the libraries should be constructed from the first-round captured PCR amplicons.
- I could have used only 1/3 of the DNA for Mme I digestion, since only 1/3 of the digested DNA was enough for the ligation. In other words, this protocol need only ~300ng of captured PCR amplicons.
- Two additional PCR cycles are enough.
- I need to do PAGE size selection on the ligation-PCR products. The captured PCR amplicons don't need to be size selected.