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==Bisulfite sequencing for Oct4, Nanog, and LMNA promoter methylation== * Measuring the methylation at several CpGs in control genes: Oct4, Nanog, and LmnA. * The data can be used as control of methylation at pluripotent genes * As requestd by Guanghui, I will primers previously published in (Mol Biol Cell. 2007 May; 18(5): 1543β1553. doi: 10.1091/mbc.E07-01-0029.) ===Primers=== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Name''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''Length''' |- | bs-Oct4-5-F||AATAGATTTTGAAGGGGAGTTTAGG||184bp |- | bs-Oct4-5-R||TTCCTCCTTCCTCTAAAAAACTCA|| chr6:31,140,029-31,140,212 |- | bs-Nanog2-F||GAGTTAAAGAGTTTTGTTTTTAAAAATTAT|| 294bp |- | bs-Nanog2-R||TCCCAAATCTAATAATTTATCATATCTTTC|| chr12:7,940,792-7,941,084 |- | bs-LMNA-F||GAAGGGTGAGTTATATTGATGGGTAT||370 |- | bs-LMNA-R||ACTCTTAAAAAAACAATCCCAAATC|| chr1:156,084,071-156,084,439 |} ===PCR=== biscvt DNA (Conc by QUBIT): #1 HGPS72 - 202.57 ng/ul #2 H9 <resolve H9 vs BJ mix-up> - 47.21 ng/ul #3 BJ fib - 46.01 ng/ul #4 BJiPS #3 - 95.62 ng/ul #5 HGPSiPS #4 - 48.93 ng/ul #6 HGPSiPS #2 - 65.92 ng/ul #7 BJiPS #6 - 26.06 ng/ul Template (10ng/ul): 1ul F primer (25uM) : 1ul R primer (25uM) : 1ul H20 : 17ul 2xiQ MM : 20ul ----------------------- 40ul Samples Setup: N1 N2 N3 N4 N5 NTC L1 L2 L3 L4 L5 NTC O1 O2 O3 O4 O5 O6 O7 NTC O - Oct4, L - LmnA, N - Nanog Program: 96C 3min 95C 30s 58C 45s 72C 45s Go to 2 for 34x 72C 5min 15C forever Purify by 1x Ampure beads. [[File:BisPCR_progeria_first_round_rt_pcr.PNG | 300px]] [[File:ZhangLab_2_2010-11-16_bis_PCR_progeria_2.jpg | 600px]] * Quantification by Nanodrop * Purification by E-Gel 2% [[File:ZhangLab_2_2010-11-16_E-Gel_ss_bisPCR_amplicons.jpg | 600px]] * Purification by 1x Ampure Beads ===Blunt-vector ligation=== '''Quick blunting Kit''' '''x 18rxn mix''' DNA 38ul - 10X blunting buffer. 5ul 90ul dNTP mix (1mM) 5ul 90ul Blunting Enzyme Mix 2ul 36 ------------------------------------------ - aliquot 12ul of reaction mix into PCR tube, - add 38ul of DNA - incubate at room temperature for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB. '''PAGE Gel''' [[File:ZhangLab_2_2010-11-17_bisPCR_progeria_blunt_ended_DNA.jpg|300px]] '''Reamplification with Phusion HF for 10 cycles''' [[File:BisPCR_progeria_second_round_rt_pcr.PNG | 300px]] * Purification with 0.7x Ampure beads * PAGE Gel [[File:ZhangLab_2_2010-11-17_bisPCR_progeria_re-amped_ended_amplicons.jpg | 300px]] * Concentrate O1-O7 using concentrator. *Invitrogen Zero-blunt PCR Cloning kit (Invitrogen) *Set up ligation reaction in PCR tubes on cold block: Sam's protocol (Invitrogen manual protocol) Mix O Mix N Mix L PCR product of seq. lib 2.0 2.3 0.8 10X Ligation buffer 1.0 1.0 1.0 pCR-Blunt (25ng/uL) 0.5 0.5 0.5 RT-PCR grade H2O 5.5 5.2 6.7 (Red cap)T4 DNA ligase (4U/uL) 1.0 1.0 1.0 ---------------------------------------------------------------------- 10.0 10.0 10.0 *Incubate at 16C for 1hr using PCR thermocycler. Perform the transfection using the ligation product as soon as possible. ===Heat-shock transfection=== *Equilibrate water bath to 42C. *Bring the SOC medium, LB plates (w/ antibiotics - Kanamycin 50ug/mL) to RT for 30min. *Thaw the TOP10 E.coli competent cell vial on ice. *Transfer '''1uL''' ligation reactions into the vial of competent cells. Mix by gentle stiring using tips. ('''Mixing by repeat pipetting is prohibited in the protocol'''). *Set up competent cell vials in the foam-board holder (for water bath). Incubate the vials on ice for 30 mins. *Adjust the timer: set up 30sec and 2min. *Heat shock the cells for 30 seconds at 42 C without shaking. Immediately transfer the vials on ice for 2min. *Add 250ul of SOC medium into each vial (drop the SOC solution slowly throw the tube wall). *Shake the vials horizontally at 37C for 1 hour using vortex on 3rd floor at the lowest speed. *During the 1hr incubation. *After 1hr shaking, plate '''50uL''' from each transformation vial on agar plates (w/ Kanamycine). **There is no need of doing blue-white screening using X-gal, because the pBlunt plasmid has selfligation-proof design. *Incubate plates at 37C for about 20hr. <6pm 11/18/10 -> 4pm 11/19/10> '''Pick up 10 colonies per sample''' *Prepare LB medium with 10% glycerol and 35ug/ml Kanamycin. *200ul per well [[File:Bis-PCR Progeria - plate layout.png]]
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