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Kun:LabNotes/CpgSeq/2010-6-8
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===Removal of amplification adaptors=== To 75ul ss-amplicon, add 5ul USER enzyme, 37C 1h; Add 10ul 10x Dpn II buffer, 5ul 100uM RE-DpnII-V6 guide oligo, 94C 2min -> 37C 3min -> add 5ul Dpn II -> 37C 2h -> add 5ul USER, 10ul 10x buffer, 85ul H2O -> 37C 2h -> 75C 20min -> purified with two Qiaquick column: 42ng/ul x 80ul = 3.4ug [[Image:2010-06-11-DMR220k-probe-prep.png|300px]] M1: 100bp lader (2uL); M2: 10bp ladder (1ul) A: Expansion PCR product, 1ul; B: Production PCR product, 1ul; C: Lambda exo digested DNA, 2ul; D: USER/DpnI digested DNA, 2ul Note: based on the gel, adaptor removal was ~70% complete. -> perform PAGE size selection on 2ug DNA with a 6% TB-Urea 2D gel. Resuspended the DNA in 30ul H2O. Quantification with PAGE gel. [[Image:2010-06-11-DMR220k-gel-quant.png|400px]] LM: Invitrogen Low Mass ladder; A: DMR220k PAGE purified probes; B: DMR220k unpurified probes; Yield: 21ng/ul (445nM) x 30ul = 630ng. I also diluted the remaining digested product in an 1:2 ratio, and quantified the two bands with PAGE gel. The completely digested band is 10.5ng/ul. The half digested band is 1.5ng/ul. Therefore the digested efficiency is 88%
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