Kun:LabNotes/CpgSeq/2010-6-8

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Probe prep for the DMR220k set[edit]

          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[edit]

                               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[edit]

   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[edit]

  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[edit]

  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
  File:2010-06-11-DMR220k-probe-prep.png
  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.
  File:2010-06-11-DMR220k-gel-quant.png
  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)[edit]

  • 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.
    File:2010-06-11-DMR220k-Capture-PCR-curves.png File:2010-06-11-DMR220k-Capture-PCR-PAGE.png
    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:
  1. 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??
  2. 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:
    1. Annealing temperature was not optimal.
    2. Probe concentration was over-estimated.
    3. The quality of Jurkat DNA was better than BJ-iPS.
    4. [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.
  3. The no-size-selected (NSS) probes have a much lower efficiency.