Alice:LabNotes/2009-7-21
Jump to navigation
Jump to search
Test the PCR-free library construction protocol
NA12878_070809: 100ul (7.08ng/ul) volume were added up to 100ul total with ddH2O (required by Covaris) Pos. CTRL: 107bp spacer (30ng/ul)
End repair (Enzymatic End-repair Mix)
PGP5 Pos. CTRL Sonicated DNA 100ul 25ulDNA + 75ul of H2O dNTP 5ul 10x buffer 12ul Enzyme mix 5ul Incubate at RT for 30-min Purified with QiqQuick columns, eluted with 38ul EB
Nanodrop: NA12878: 20.5ng/ul positive control: 7ng/ul
A-tail addition
Blunt-ended DNA 35ul 10X Klenow buffer 5ul 1mM dATP 10ul Klenow fragment (exo-) 1ul 37C 30min, purified with MinElute columns, eluted with 12ul EB.
TA ligation
- Prepare 20uM adaptor oligo duplex.
100uM A_adaptor_t 20ul 100uM A_adaptor_b 20ul 10x T4 ligase buffer 5ul H2O 55ul 94C 3min -> cool down to 20C at the rate of 0.05C/sec.
- TA ligation:
- Adaptor/insert ratio should be ~10:1, which would be roughly 2ul per 400ng sonicated DNA.
PGP5 Pos. CTRL Neg. CTRL DNA (~500ng) 11ul 11ul 11ul H2O 2x QuickLigase buffer (NEB) 16ul 16ul 16ul 20uM Adaptor oligo mix 3ul 3ul 3ul T4 DNA QuickLigase (NEB) 2ul 2ul 2ul extra ATP 5ul 5ul 5ul Incubate at RT for 15 minutes. Purified with MinElute columns, eluted with 20ul EB
PCR of sequencing library
x3 Template 5ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 2ul Solexa_PCR_lo_PE (10uM) 2ul H2O 41ul 50X SYBG I 0.2ul
98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 11 cycles of (98C 10sec -> 72C 15 sec)-> 72C 3min -> 15C hold. File:ZhangLab 2 2009-07-21 21hr 02min.jpg
Qiaquick column purification, elute in 30ul EB Nanodrop: 274ng/ul (260/280: 1.91)
Egel size selection at around 200-225bp gel quantification: 2.5ng/ul (estimated as 10ng/ul) Qubit: 27ng/ul
sequencing run
' | Sample | Amount loaded | Library conc. |
Lane 1 | phiX | 3pmole | |
Lane 2 | phiX | 4pmole | |
Lane 3 | NA12878 | 4pmole | 10nM |
Lane 4 | Hues6ES-DSp | 5pmole | 38nM |
Lane 5 | HUES6ES-tRp | 5pmole | 28nM |
Lane 6 | Hybrid1-EB | 5pmole | 606nM |
Lane 7 | Hybrid2-ES | 5pmole | 395nM |
Lane 8 | Hybrid2-EB | 5pmole | 303nM |