Kun:LabNotes/ExonomeSeq/2008-1-24
Exp.1 Library construction on the new capturing reaction started on 01/16/2008
Mme I digestion on the 2nd round PCR amplicon
500bp+ 2nd PCR all size 2nd PCR 50ul 50ul 32mM SAM 0.5ul 0.5ul Mme I 2U/ul 4ul 4ul 37C 1h -> 75C 20min -> 4C hold. Purify with Bioneer columns, then PAGE size selection. Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis.
USER enzyme digestion
Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h.
End repair
Set up end repair reaction (with NEB quick blunting kit)
USER DNA 19ul 10X buffer 2.5ul 1mM dNTP 2.5ul Enzyme mix 1ul RT for 30min -> 75C 10min
SolN6 primed synthesis
SolF/Rn6 DNA 10ul 10X Klenow buffer 5ul 100uM SolFn6 1ul 10mM dNTP 2ul H2O 30ul
94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB.
Ligation
USER DNA 15ul 50uM Solexa_1 2ul 50uM Solexa_2 2ul 2X Quick ligation buffer 20ul Quick ligase 1ul mix well -> room temperature for 20min -> 75C 10min Purified with MinElute columns.
Nick-translation
DNA 15ul 10X ThermoPol Buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul 8U/ul Bst pol 1ul 65C 25min -> place on ice
PCR
Nick-translated DNA 5ul SolN6 primed DNA 5ul 2X iProof master mix 25ul 25ul 100uM Solexa_PCR_up 0.25ul 0.25ul 100uM Solexa_PCR_lo 0.25ul 0.25ul H2O 19.3ul 19.3ul 50X SYBG I 0.2ul 0.2ul 98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
Exp.2 Probe prep for Cosmic3760, CpG28k and CES27k
PCR (with NEB Taq master mix)
Cosmic3760 CpG28k CES27k x1 x25 Cosmic3760(20nM) 0.1ul 2.5ul CpgCES (20nM) 2.5ul 2.5ul 2x Taq master mix 50ul 1250ul 1250ul 1250ul 100uM AP1V41U 0.4ul 10ul 10ul pAP1V6U 10ul 100uM pAP2V4 0.4ul 10ul 10ul phosAP2V6 10ul H2O 49.3ul 1230ul 1230ul 1230ul
94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold
Ethanol precipitation.
Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min.
Purify with Qiaquick columns. Yield: Cosmic3760=53ng/ul x 80ul; Cpg28k=33ng/ulx80ul; CES27k=33ng/ul x 80ul.
Added 4ul USER enzyme, 37C 2h.
Add 90ul H2O, 20ul 10X DpnII buffer, 10ul DpnIIV4/5ul DpnIIV4/5ul RE-DpnIIs, 94C 5min -> slowly cool down to 37C -> add 10ul Dpn II -> 37C 2h -> add 5ul USER -> 37C 2h -> 75C 10min -> 4C hold.
I took 100ul Cosmic3760 and 100ul CpG28k out after 2-hour digestion. Did phenol/chloroform extraction, then PAGE purification. The palettes were resuspended in 10ul phosphorylation mix (1X T4PNK buffer, 1mM ATP, 1U/ul T4 PNK), 37C 10min. Then diluted to 20ul with ddH2O.
The remaining digestions were left until the reactions were completed. I heated the reactions at 95C for ~10min. Then did another round of PAGE on 100ul reactions on 01/26/08.