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===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.
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