Kun:LabNotes/CpgSeq/2010-6-8: Difference between revisions

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Line 17: Line 17:
   H2O:                          96ul
   H2O:                          96ul
   94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 16 -> 72C 3min -> 15C hold   
   94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 16 -> 72C 3min -> 15C hold   
   Purify the amplicons with two Qiaquick columns, eluted with 40ul EB.
  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===
===Production PCR===
     10nM 1st round amplicon       5ul
     10nM 1st round amplicon     0.2ul
     2x Kapa SYBG qPCR Master Mix  50ul
     2x Kapa SYBG qPCR Master Mix  50ul
     100uM pAP1V61U              0.4ul
     100uM pAP1V61U              0.4ul
Line 25: Line 32:
     H2O                          44ul
     H2O                          44ul
     ===================================
     ===================================
                                 100ul x 16
                                 100ul x 32
     95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 14 -> 60C 2min -> 15C hold.
     95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold.
     Ethanol precipitation.
     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


===Digestion and purification===
    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.

Latest revision as of 01:05, 15 June 2010

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.