Kun:LabNotes/ExonomeSeq/2008-1-8
Exp.1 Perform 2nd round PCR on the PAGE purified 450bp band from the previous capturing experiment.[edit]
dNTP dUTP Template: 5ul 5ul 10X PCR buffer: 10ul 10ul 10mM dNTP: 2ul 2ul 1mM dUTP: 0ul 1ul 100uM AmpFSolV6.2 0.4ul 0.4ul 100uM AmpRSolV6.2 0.4ul 0.4ul 50X SYBG I: 0.4ul 0.4ul JumpStart Taq: 2ul 2ul H2O: 80ul 79ul
94C 3min -> (94C 30sec -> 72C 1min) x 10cyc -> 72C 3min -> 15C hold. Purify 50ul with Bioneer columns. dNTP: 31ng/ul x 30ul dUTP: 30ng/ul x 30ul
Exp.2 Make sequencing library by USER digestion and ligation[edit]
15ul dUTP amplicon -> add 1.5ul USER enzyme -> 37C 1h -> set up ligation.
Exp. 3 Make sequencing library by random priming[edit]
SolF/Rn6 N6 DNA 5ul 5ul 10X Klenow buffer 2.5ul 2.5ul 100uM SolFn6 0.25ul 0ul 100uM SolRn6 0.25ul 0ul 1mM N6 0ul 0.5ul 10mM dNTP 1ul 1ul H2O 14ul 14ul
94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> purified with MinElute columns.
Setup ligation for the N6 reaction.
Exp.4 Perform another round of exon capture experiment[edit]
Sheared NA10835(~300ng/ul) 3ul E55kv1 probes 107nM (01/07/08,purified) 17ul 10X Ampligase buffer 2.25ul
95C 10min -> 60C 40h -> add 2.5ul NSL mix (Stoffel 1U/ul, AmpLigase 0.5U/ul, dNTP 5uM) -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min
Not sure I added the right NSL mix or the one for SNP . So I re-started the circularization reaction: 95C 5min -> 60C 20h -> add 2.5ul NSL mix -> 60C 1h -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold
Exp.5 Another round of probe prep for E55kv1 and CES22k #5[edit]
PCR (with NEB Taq master mix)
x1 x25 E55k1(20nM) 0.1ul 2.5ul CES22k #5(100nM) 0.5 2x Taq master mix 50ul 1250ul 1250ul 100uM pAP1V6U 0.4ul 10ul 10ul 100uM phosAP2V6 0.4ul 10ul 10ul H2O 49.3ul 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 50ul with MinElute columns. Measure concentration:
- CES22k #5: 98ng/ul x 20ul
- E55kv1: 71ng/ul x 20ul
Added 2ul USER enzyme, 37C overnight. Added 20ul dH2O, 5ul 10X Dpn II buffer, 2ul RE-DpnIIs guide oligo. 95C 10min -> turn off the heater, let the reaction slowly cool down in ~15 minutes -> add 4ul Dpn II -> 37C 1 hour -> add 4ul USER enzyme -> 37C 2 hour.
Clean up the remaining 150ul ssDNA with G25 column, add 10ul USER enzyme, 37C 2h -> add 20ul Dpn II buffer, 6ul RE-Dpn IIs guide oligo. 95C 10min -> turn off the heater, let the reaction slowly cool down in ~15 minutes -> add 12ul Dpn II -> 37C 1 hour -> add 8ul USER enzyme -> 37C 2 hour.
50ul of the first batch digestion + 50ul of the second batch digestion -> PAGE purification with one 2D TBU gel.
Phenol/chloroform extraction of the remaining 150ul second batch digestion. -> PAGE purification of 50ul with 2D TBU gel.
Yield:
- E55kv1, no phenol extraction: 5.1ng/ul => 180nM x 30ul
- E55kv1, phenol extraction: 7.8ng/ul => 275nM x 30ul
- CES22k #5, no phenol extraction: 6.4ng/ul => 246nM x 30ul
- CES22k #5, phenol extraction: 9.3ng/ul => 358nM x 30ul