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Kun:LabNotes/CpgSeq/2010-6-8
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==Probe prep for the DMR220k set== Library name OLID Part # DMR220k_a 0284601 G4446A DMR220k_b 0284591 G4446A DMR220k_c 0284581 G4446A DMR220k_d 0284571 G4446A *Resuspend the oligos in each tube with 500ul nuclease-free H2O. ===Expansion PCR=== x 4 DMR220k_a (20nM) 1ul DMR220k_b (20nM) 1ul DMR220k_c (20nM) 1ul DMR220k_d (20nM) 1ul 2x iTaq master mix: 100ul 100uM AP1V6: 0.8ul 100uM AP2V6: 0.8ul H2O: 96ul 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 16 -> 72C 3min -> 15C hold The library was slightly over-amplified. Repeated the experiment with 13 cycles. Purify the amplicons with four Qiaquick columns, eluted with 40ul EB. Yield: 20ng/ul x ~150ul = ~3ug. Concentration = 20ng/ul /(146bp*660Da/bp) = 208nM Take 20ul, add 396ul EB -> 10nM ===Production PCR=== 10nM 1st round amplicon 0.2ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul H2O 44ul =================================== 100ul x 32 95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold. Ethanol precipitation; re-purified with four Qiaquick columns, each eluted with 40ul EB. Yield: 214ng/ul x 160ul = 34ug ===Lambda exo digestion=== To 100ul (21ug) Qiaquick amplified amplicon, add 15ul 10x buffer, 10ul Lambda exo, 25ul H20 -> 37C 1h -> purified with two Qiaquick columns, each eluted with 40ul EB. Yield: 76ng/ul x 80ul = 6.1ug ===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% ===Test the probes by performing the capture experiment on BJ-iPS (Salk)=== *Dinh did the bisulfite conversion and provided me the DNA. *I will also do one capture reaction using the digested probes without size selection (N.S.S.). *I will include a capture reaction using the Cpg97kA probe set on Jurkat DNA (not enough BJ-iPS DNA). The probe concentration is 15ng/ul (310nM) *Template DNA: ~300ng; probe:target ratio=200:1. A B C BJ-iPS(247ng/ul) BJ-iPS(247ng/ul) Jurkat(271ng/ul) Template DNA 1.5ul 1.5ul 1.5ul Probe DMR220k 8.2ul DMR220k(NSS) 16ul Cpg97kA 2.7ul 10X buffer 2.0ul 2.0ul 2.0ul H2O 8.3ul 0.5ul 13.7ul 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold. Note on dNTP concentration: based on Li et al GR 2009 (PMID 19525355), the optimal dNTP concentration is 10X of the concentration required to circularize 100% of template molecules. For 300ng of input DNA, 1X concentration is equivalent to: 300ng / 3pg (one copy of haploid genome) x 220,000 (probes) x 200 (insert length) / 6E23 = 7.3E-12 mole 10X concentration is 73 pmole. Since we are adding 2ul of SLN mix, the dNTP concentration for the SLN mix should be 37uM. Here are used a slightly high concentration of 50uM to be conservative. PCR 2X iProof Mastermix 50ul AmpF6.3Sol (100uM) 0.2ul AmpR6.3Sol (100uM) 0.2ul 50X SYBG I 0.4ul H2O 40ul Captured DNA 10ul 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 8 -> (98C 10S -> 72C 20S) x 8 ->72C 3 min -> 15C hold. [[Image:2010-06-11-DMR220k-Capture-PCR-curves.png|300px]] [[Image:2010-06-11-DMR220k-Capture-PCR-PAGE.png|300px]] Purified each amplicon with one Qiaquick column, eluted with 40ul EB. Yield: A: 16ng/ul x 40ul B: 8ng/ul x 40ul C: 23ng/ul x 40ul I did a size selection on the A amplicon using the Invitrogen 2% Size-select gel, and gave the DNA to Alan for end-sequencing using SolSeqv6.3 primer. *Discussion: #DMR220k probe set was designed to have the same PCR amplicon length (235bp for insert+H1+H2; 329bp plus Amp[F/R]6.3Sol). However, I saw a smear in amplicon A. I've double-check the OLIDs on the tubes and the sequences uploaded. They are all correct. The sizes of the probe library with and without the AP[1/2]V6 adaptors are also expected. The Cpg97k library was smaller (102nt without the adaptors and 140nt with the adaptors). Therefore the smear was not due to the probe design and synthesis. I think this is most likely due to the non-specific capture. In the size-select gel, the smear looked a lot sharper. Perhaps it's due to the combination of over-loading in PAGE gel and slightly over-amplification?? #The real-time curve A didn't come up much earlier than C, suggesting that the overall capturing efficiency was lower then the Cpg97kA set. This could be due to the follow reasons: ##Annealing temperature was not optimal. ##Probe concentration was over-estimated. ##The quality of Jurkat DNA was better than BJ-iPS. ##[dNTP] was too low for this probe set. Note that the conclusion drawn in the GR paper was based on a probe set that has roughly 10x less probes. Will definitely try 200uM in the next experiment. # The no-size-selected (NSS) probes have a much lower efficiency.
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