Kun:LabNotes/CpgSeq/2008-3-18: Difference between revisions

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**[[Media:cpgIslandExt_250bp_1N_chr21.probes.xls|Chr21 probes]]
**[[Media:cpgIslandExt_250bp_1N_chr21.probes.xls|Chr21 probes]]
**[[Media:cpgIslandExt_250bp_1N_chr22.probes.xls|Chr22 probes]]
**[[Media:cpgIslandExt_250bp_1N_chr22.probes.xls|Chr22 probes]]
  77/94 are on-target capture, 16 are primer concatemers, 1 seems to be cloning artifact.
  [[Image:Cpg031808-dist.png|400px]]
==Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)==
==Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)==
     Template                    5ul
     Template                    5ul
Line 259: Line 262:
     I think it is due to the 85C head inactivation step, which denatured DNA. So what I was see are  
     I think it is due to the 85C head inactivation step, which denatured DNA. So what I was see are  
     ssDNA or partially annealed ds/ssDNA.
     ssDNA or partially annealed ds/ssDNA.
  [[Image:2008-04-11-Cpg-Jurkat-2nd-PCR-MmeI-cut.png|300px]]
==Perform another PCR on the remaining PCR amplicon (27ng/ul)==
==Perform another PCR on the remaining PCR amplicon (27ng/ul)==
     Template                    4ul
     Template                    4ul
Line 278: Line 283:
   H2O                        10ul
   H2O                        10ul
   37C 90min 4C hold.
   37C 90min 4C hold.
  Qiaquick purification -> 6% TBE PAGE size selection
  The DNA migration pattern was still not right. Looks like Mme I didn't make any cut at all.
  Repeat the digestion by adding 36ul H2O, 5ul 1mM SAM, 4ul Mme I, 5ul 10X NEBuffer 4.
  37C 4h.
  [[Image:2008-04-13-Cpg-Jurkat-MmeI-Size-selection.png|200px]]
====End repair (Epicentre End-it repair kit)====
  Resuspended the pallet in 30ul ddH2O, add 3ul USER enzyme, 37C 4h.
  Add 5ul 10x Buffer, 5ul dNTP mix, 5ul ATP, 1ul Enzyme mix.
  RT overnight.
  Purified with MinDlute kit. 16ng/ul x 10ul
====Blunt-end ligation====
  25ng/ul end-repaired DNA  10ul
  100uM Solexa_1 pair      0.5ul
  100uM Solexa_2 pair      0.5ul
  2X Quick ligase buffer    15ul
  Water                      3ul
  Quick Ligase (NEB)          1ul
  RT 10min -> Purified with Qiaquick column.
  PAGE size selection, ethanol precipitation
  [[Image:2008-04-15-Sol-adaptor-ligation-size-selection.png|300px]]
===Nick-translation===
  DNA                    15ul
  10X ThermoPol Buffer    2ul
  10mM dNTP            0.4ul
  1mg/ml BSA              2ul
  8U/ul Bst pol          1ul
  65C 25min -> place on ice
===PCR===
    Nick-translated DNA    10ul
    2X iProof master mix  100ul
    100uM Solexa_PCR_up      1ul
    100uM Solexa_PCR_lo      1ul
    H2O                  87.3ul
    50X SYBG I            0.8ul
    98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold
  [[Image:2008-04-17-Solexa-lib-PCR.png|300px]]
==Perform another round of PCR on the 04/10/08 amplicon (27ng/ul)==
    Template                    5ul
    10X Platinium Taq buffer  40ul
    50mM MgCl2                12ul
    100uM AmpF6.2SolH        1.6ul
    100uM AmpR6.2SolH        1.6ul
    10mM dNTP                  8ul
    1mM dUTP                    2ul
    50X SYBG I                1.6ul
    H2O                      326ul
    JumpStart Taq(5U/ul)        4ul
    split into 4x100ul
    94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold.
    Purified 4X100ul amplicons with 2x Zymo DNA concentrator-25 columns.
    Eluted DNA from each column with 30ul EB buffer.
    Yield: 47ng/ul x 60ul.
    [[Image:2008-04-12-Cpg-Jurkat-3rd-PCR-amplicons.png|200px]]
====Mme I digestion====
  DNA                        30ul x 47ng/ul
  10X NEBuffer 4:              5ul
  1mM SAM                    2.5ul
  Mme I (2U/ul)                5ul
  H2O                        10ul
  37C 1h.
   Qiaquick purification -> 6% TBE PAGE size selection
   Qiaquick purification -> 6% TBE PAGE size selection

Latest revision as of 01:17, 18 April 2008

Exp. 1 Cpg capture with a new batch of bisulfite treated Jurkat DNA from 03/17/08[edit]

                      Sigma x2     Zymo x 2
  Template              7            3
  10X buffer(fresh)     1            1
  Cpg28k probe(218nM)   2            2
  H2O                   0            4
  Tubes:
  A: Sigma, dNTP 10nM
  B: Sigma, dNTP 50uM
  C: Zymo, dNTP 10nM
  D: Zyno, dNTP 50uM

Exon capture control

  GM20431 131ng/ul      7  (10/12/2006)        
  10X buffer(fresh)     1           
  E55kv1 probe(220nM)   2  (01/12/2008)        
  H2O                   0            

94C 10min -> 60C 10min -> add 1ul NSL mix (Stoffel 2U/ul, Ampligase 0.5U/ul, dNTP 10nM or 50uM) -> 60C 1h -> 45 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul Exo I&III, 37C 2h -> 94C 5min -> 4C hold.

PCR[edit]

   Template                 12ul
   2X iProof Mastermix     100ul
   100uM AmpF6.2           0.8ul
   100uM AmpR6.2           0.8ul
   50X SYBG I              0.8ul
   H2O                      85ul
   Split into two tubes.
   98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 15 
           -> 72C 5 min -> 15C hold.
  File:2008-03-21-Cpg-E55kv1-captured-PCR.png
  A: Sigma, dNTP 10nM
  B: Sigma, dNTP 50uM
  C: Zymo, dNTP 10nM
  D: Zymo, dNTP 50uM
  E: E55kv1, dNTP 10nM
  F: E55kv1, dNTP 50uM
  To test whether some of the high MW bands are concatemers, 
  I did Alu I digestion on the MinElute purified amplicons.
  8ul DNA + 1ul 10X NEBuffer2 + 1ul Alu I -> 37C 30min -> 75C 10min. 
  File:2008-03-21-capured-amplicon-AluI-cut.png

PAGE purification of amplicon D, F, and Cosmic C(03/17/08)[edit]

 File:2008-03-21-PAGE.png
 Resuspended the pellets in 15ul ddH2O.

Blunt end cloning[edit]

  PAGE purified amplicons:  3.5ul
  10X ligation buffer:      0.5ul
  Zero Blunt vector:        0.5ul
  T4 DNA ligase:            0.5ul
  16C 1h -> 4C overnight.

Colony PCR & sequencing[edit]

I pick 16 colonies from the CpG plate, 8 from the Cosmic plate and 8 from the E55kv1 plate.

PCR:

   2X TaqMaster mix            15ul
   10uM M13 F/R primer mix    0.6ul
   H2O                       14.4ul
   94C 2min -> 35 cycles of (94C 30sec -> 52C 30sec -> 72C 30sec) -> 72C 3min 

Check with Argarose gel, 4ul amplicons per lane, the ladder is Invitrogen Low Mass ladder (4ul).

   Cpg #1:  Cpg A1         Cosmic #1: Cosmic A1
   Cpg #2:  Cpg B1         Cosmic #2: Cosmic B1
   Cpg #3:  Cpg C1         Cosmic #3: Cosmic C1
   Cpg #4:  Cpg D1         Cosmic #4: Cosmic D1
   Cpg #5:  Cpg E1         E55kv1 #1: E55kv1 A1
   Cpg #6:  Cpg F1         E55kv1 #1: E55kv1 A1
   Cpg #7:  Cpg H1         E55kv1 #1: E55kv1 D1
   Cpg #8:  Cpg B2         E55kv1 #1: E55kv1 E1

Analysis of sequencing results[edit]

Cosmic amplicons[edit]

             Insert(incl. H1&H2)   Target           Note
  Cosmic #1:      235bp         FRMD4A exon   1 mismatch in H2
  Cosmic #2:       65bp, contains AP1V6 and AP2V2 sequences, seems to due to chimerism in probe prep
  Cosmic #3:      226bp         RIMS2 exon    100% match
  Cosmic #4:     Similar to #2, also has amplification adaptors
 
  Raw sequences of #2
  >Cosmic2-T7.ab1
  XXXXXXXXXXGCAGCTGAATAGGAACGATGAGCCTCCAACACCCAATCTT
  AAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCAGTCTACAACAGTGCT
  CTTCCAGTCTACCTAGCCTCATGCGTATCCGATCCAGAAGGCCAGAAATG
  GCAGTGTCGGACCTCGATAACATCTGCAGCTGAATAGGAACGATGAGCCT
  CCAACACCCAATCTTAAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCA
  GTCTACAACAGTGCTCTTCCAGTCTACCTAGCCTCATGCGTATCCGATCC
  AGAAGGCCAGAAATGGCAGTGTCGGACCTCGATAACATCTGCA   
  Raw sequences of #4
  >Cosmic4-T7.ab1
  XXXXXXXXXXXXTGCAGATGTTATCGAGGTCCGACAAACCAATTGCACAG
  CACACGATCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTAC
  ACTAAGAGCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTA
  TTCAGCTGCAGATGTATCGAGGTCCGACAAACCAATTGCACAGCACACGA
  TCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTACACTAAGA
  GCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTATTCAGCT
  GC

Cpg #1[edit]

The capturing sequences were mapped to the probe targeting the CpG island at chr22:38387887-38388790. The insert is 192bp, same as the expected size.

I did blast pair-wise alignment between the insert and the targeted genomic sequence.

  Query  34   CGTCGTTTTAGTTGGAATAGTTTTAAGTATAAGTCGTCGTCGGTGGAGTATGAGTTTTTG  93
              ||||| |  || ||||| ||  | ||| | ||| || |||||| |||| ||||||   ||
  Sbjct  187  CGTCGCTCCAGCTGGAACAGCCTCAAGCACAAGCCGCCGTCGGCGGAGCATGAGTCCCTG  246
  Query  94   TTTTTTGAGGAGCGCGGCGGCGGCGTTCGGGTTTGCGAGGTTGTCGCGGACGAGGGGTCG  153
               | | || |||||||||||||||||  ||||| |||||||||| ||||||||||||| ||
  Sbjct  247  CTCTCTGCGGAGCGCGGCGGCGGCGCCCGGGTCTGCGAGGTTGCCGCGGACGAGGGGCCG  306
  Query  154  TCGTGGGTCGTATTTTTGTATATTTTATACGTTTATTACGTTTATTATGGGTTTTATTTG  213                     
              || ||| || |    || | |    | |||   |  || || || | |||    || ||
  Sbjct  307  CCGCGGGCCGCACCCCTGCACACCCCACACGCCCACCACATTCATCACGGGCCCCATCTG  366
  Query  214  GTGTATCGTTATCGTTATTATCGTCGGACGTTGTTTTTCGATAATAGGGATTTGGTGGAT  273
              | | | ||  | ||  |  | || |||||| |||   |||| || ||||| | |||||| 
  Sbjct  367  GCGCACCGCCACCGCCACCACCGCCGGACGCTGTCCCTCGACAACAGGGACTCGGTGGAC  426
  Query  274  TTGGTCG  280
               ||| ||
  Sbjct  427  CTGGCCG  433

Cpg #2[edit]

The capturing sequences were mapped to the probe targeting the CpG island at chr20:61,136,225-61,137,000. The insert is 182bp, same as the expected size.

I did blast pair-wise alignment between the insert and the targeted genomic sequence.

  Query  41   GGTGGTTTGGGTCGGTGTTTAGTTTAGGGTTTCGCGGTTAGAGTTAATTATGGCGCGTTT  100
              |||||  |||||||||| | ||   |||||  |||||  ||||  ||  ||||||||  |
  Sbjct  70   GGTGGCCTGGGTCGGTGCTCAGCCCAGGGTCCCGCGGCCAGAGCCAACCATGGCGCGCCT  129
  
  Query  101  GGTTTTTTTTAGATAAACGCGCGTTTACGTTTTTGTTGGGTTTTGGAATTAGTTTAATAT  160
              ||| |  |  |||||||||||||   |||| | || |||| ||||||||||| | |||| 
  Sbjct  130  GGTCTCCTCCAGATAAACGCGCGCCCACGTCTCTGCTGGGCTTTGGAATTAGCTCAATAC  189
  
  Query  161  TCGTTTAGACGTAGAGTGCGTGTGTTTTGTTAGGATAGACGTTATTGTTGTGTGGTGTTT  220
              || || ||||| ||||||||||||   ||  |||| |||||| |||| |||| |||| ||
  Sbjct  190  TCATTCAGACGCAGAGTGCGTGTGCCCTGCCAGGACAGACGTCATTGCTGTGCGGTGCTT  249
  
  Query  221  GAGTGTTATTAATAAGTTATAGAAGAGAAATTCGGAAATAAGGGA  265
              |||||  || ||||||  | ||||||||||| |||||| ||||||
  Sbjct  250  GAGTGCCATCAATAAGCCACAGAAGAGAAATCCGGAAACAAGGGA  294

Cpg #3[edit]

Tandem concatemers of AP1V4 and AP2V2, plus half of the probes for c20:19945840w. I think this is due to the chimerism of the first PCR step.

Cpg #4[edit]

Similar to Cpg #3: tandem concatemers of AP1V4 and AP2V2, plus half of the probes for c20:30048155c. I think this is due to the chimerism of the first PCR step.

Cpg #5[edit]

The first 22-235bp are AT rich sequence that I haven't been able to mapped to the genome. 236-295bp can be mapped to multiple positions in hr19:42463268-42490100, which contains mostly SST1 satellite repeats. I think this is due to probe design issues.

Cpg #6[edit]

The capturing arms were mapped to the probe targeting c22:38387974w. Insert is 172bp, which is what I expected. However, I can't map the insert to the target, which is probably due to problem with C:T mismatches.

I did the alignment manually, which turned out to be successful. The reason Blast didn't pick up the alignment was due to sequencing errors in homopolymer runs.

Cpg #7[edit]

The capturing sequences were mapped to the probe targeting the CpG island at chr19:41485375-41485793. The insert is 211bp, same as the expected size.

I did blast pair-wise alignment between the insert and the targeted genomic sequence.

 Query  44   AGGAAGGTTAGTTTTTGAGAGATAGATATGTTTGTTTTATTTTTTTGTCGGTTTTTTTTT    103
             |||||||  ||  ||||||||| ||| ||| |||    ||    ||| ||| ||  || |
 Sbjct  567  AGGAAGGCCAGCCTTTGAGAGACAGACATGCTTGCCCCATCCCCTTGCCGGCTTCCTTCT  508
 
 Query  104  TCGTTTTTGCGTCGAGTTGTGGTTATATTTTTCGATGAGGGTAGAGGGCGATAGGCGTGA  163
              |||   ||||||||| ||||| || |||| |||||||||| ||||||||| ||||||||
 Sbjct  507  CCGTCCCTGCGTCGAGCTGTGGCTACATTTCTCGATGAGGGCAGAGGGCGACAGGCGTGA  448
 
 Query  164  TAATTATTTTTTTGAAGTTTTGCGGGTATTTTTTTGCGGGTAGACGATGAGCGTTTGGGA  223
              ||  |  || |||||| | |||||| |   |  ||||||| |||||||||||  |||||
 Sbjct  447  CAACCACCTTCTTGAAGCTCTGCGGGCACCCTCCTGCGGGTGGACGATGAGCGCCTGGGA  388
 
 Query  224  GGTCGTTGTTTTTGGTTGGGGAGCGTTCGTTTGGATTTAGTTTAGTAAAGAGGTTGTTTC  283
             || ||||||  |||||||||||||| |||| |||||  ||  ||| ||||||| || | |
 Sbjct  387  GGCCGTTGTCCTTGGTTGGGGAGCGCTCGTCTGGATCCAGCCTAGCAAAGAGGCTGCTCC  328
 
 Query  284  GGATGGGGAGGG  295
             ||||||||||||
 Sbjct  327  GGATGGGGAGGG  316

Cpg #8[edit]

The capturing sequences were mapped to the probe targeting the CpG island at c19:42463450c.

I did blast pair-wise alignment between the insert and the targeted genomic sequence.

 Query  51   TGGGTCGTGTGGTCGGTTGGTAGTCGTCGTTTCGGCGGTTTTAGTTTGGGGGTTTGTGAA  110
             |||||||||||| ||||||| |||||||||  | || |||  ||||||||||| ||||||
 Sbjct  429  TGGGTCGTGTGGCCGGTTGGCAGTCGTCGTCCCAGCAGTTCCAGTTTGGGGGTCTGTGAA  370
 
 Query  111  GGTTTGGGTAACGTGGGTATGGGCGTCGGATTCGTA-GGGTTTTTATTTTTTTTTTATTC  169
             ||  |||| |||||||| || ||||||| |  || | ||||||| ||    |    || |
 Sbjct  369  GGCCTGGGCAACGTGGGCATCGGCGTCGAACCCGCAGGGGTTTTCATCCCCTCCCCATCC  310
 
 Query  170  GGAGTAGTTTTTTTGTTAGGTTGGATTTAGACGAGCGTTTTTTAATTAAGGATAACGGTT  229
             |||| ||  | |||| |||| |||||  ||||||||| |    ||  ||||| |||||  
 Sbjct  309  GGAGCAGCCTCTTTGCTAGGCTGGATCCAGACGAGCGCTCCCCAACCAAGGACAACGGCC  250
 
 Query  230  TTTTAGGCGTTTATCGTTTATTCGTAGGAGGGTGTTCGTAGAGTTTTAAGAAGGTGGTTG  289
             |   ||||| | || ||  |  || |||| ||||  || |||| || |||||||||||||
 Sbjct  249  TCCCAGGCGCTCATTGTCCACCCGCAGGAAGGTGCCCGCAGAGCTTCAAGAAGGTGGTTG  190
 
 Query  290  TTACG  294
             | |||
 Sbjct  189  TCACG  185

Summary of Cpg capture[edit]

  • 5/8 amplicons were expected targets.
  • 2/8 contains mostly primer concatemers due to issues in probe prep.
  • 1/8 could not be mapped uniquely because the probe was design for a repetitive region not masked in UCSC genome draft.

I submitted another 96 clones to Agencourt for sequencing, received the data on 04/08/08[edit]

  77/94 are on-target capture, 16 are primer concatemers, 1 seems to be cloning artifact.
  File:Cpg031808-dist.png

Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)[edit]

   Template                    5ul
   10X Platinium Taq buffer   40ul
   50mM MgCl2                 12ul
   100uM AmpF6.2SolH         1.6ul
   100uM AmpR6.2SolH         1.6ul
   10mM dNTP                   8ul
   50X SYBG I                1.6ul
   H2O                       326ul
   iTaq(5U/ul)                 8ul
   split into 4x100ul
   94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 9 -> 72C 3min -> 15C hold.
   Mix all 4x100ul amplicon, purified 100ul with Qiaquick, 100ul with Zymo Concentrator-25.
   Qiaquick: 22ng/ul x 30ul
   Zymo: 28ng/ul x 15ul
   Clonewell size selection on the Qiauick purified DNA.
   Purify the remaining 200ul amplicon with Qiaquick, 34ng/ul x 30ul

Make shotgun sequencing library[edit]

Mme I digestion[edit]

  Qiaquick purified DNA 26ul x 34ng/ul
  10X NEBuffer 4:              6ul
  1mM SAM                      2ul
  Mme I (2U/ul)                5ul
  H2O                         21ul
  37C 90min -> 80C 20min -> 4C hold.
  I ran the digestion in 6% PAGE gel without purification, it turned out that 
      the DNA migration pattern was totally off, probably due to DNA:protein interaction.

Perform another PCR on the remaining PAGE size selected DNA (04/10/08)[edit]

   Template                    5ul
   2x Taq Master mix         200
   100uM AmpF6.2SolH         1.6ul
   100uM AmpR6.2SolH         1.6ul
   1mM  dUTP                   4ul
   50X SYBG I                3.2ul
   H2O                       172ul
    split into 4x100ul
   94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 8 -> 72C 3min -> 15C hold.
   Mix all 4x100ul amplicon, purified with Zymo Concentrator-25.

Make shotgun sequencing library[edit]

Mme I digestion[edit]

  Qiaquick purified DNA 26ul x 34ng/ul
  10X NEBuffer 4:              6ul
  1mM SAM                      2ul
  Mme I (2U/ul)                5ul
  H2O                         21ul
  37C 90min -> 85C 20min -> 4C hold.
  MinElute purification -> 6% TBE PAGE size selection -> the DNA migration pattern is still not right
    I think it is due to the 85C head inactivation step, which denatured DNA. So what I was see are 
    ssDNA or partially annealed ds/ssDNA.
  File:2008-04-11-Cpg-Jurkat-2nd-PCR-MmeI-cut.png

Perform another PCR on the remaining PCR amplicon (27ng/ul)[edit]

   Template                    4ul
   2x Taq Master mix         150ul
   100uM AmpF6.2SolH         1.2ul
   100uM AmpR6.2SolH         1.2ul
   1mM  dUTP                   3ul
   50X SYBG I                2.4ul
   H2O                       129ul
    split into 3x100ul
   94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold.
   Purified with Zymo Concentrator-25.

Make shotgun sequencing library[edit]

Mme I digestion[edit]

  DNA                         20ul x 29ng/ul
  10X NEBuffer 4:              4ul
  1mM SAM                    1.3ul
  Mme I (2U/ul)                4ul
  H2O                         10ul
  37C 90min 4C hold.
  Qiaquick purification -> 6% TBE PAGE size selection
  The DNA migration pattern was still not right. Looks like Mme I didn't make any cut at all.
  Repeat the digestion by adding 36ul H2O, 5ul 1mM SAM, 4ul Mme I, 5ul 10X NEBuffer 4. 
  37C 4h.
  File:2008-04-13-Cpg-Jurkat-MmeI-Size-selection.png

End repair (Epicentre End-it repair kit)[edit]

  Resuspended the pallet in 30ul ddH2O, add 3ul USER enzyme, 37C 4h.
  Add 5ul 10x Buffer, 5ul dNTP mix, 5ul ATP, 1ul Enzyme mix.
  RT overnight.
  Purified with MinDlute kit. 16ng/ul x 10ul

Blunt-end ligation[edit]

  25ng/ul end-repaired DNA   10ul
  100uM Solexa_1 pair       0.5ul
  100uM Solexa_2 pair       0.5ul
  2X Quick ligase buffer     15ul
  Water                       3ul
  Quick Ligase (NEB)          1ul
  RT 10min -> Purified with Qiaquick column.
  PAGE size selection, ethanol precipitation
  File:2008-04-15-Sol-adaptor-ligation-size-selection.png

Nick-translation[edit]

  DNA                    15ul
  10X ThermoPol Buffer    2ul
  10mM dNTP             0.4ul
  1mg/ml BSA              2ul
  8U/ul Bst pol           1ul
  65C 25min -> place on ice

PCR[edit]

    Nick-translated DNA     10ul
    2X iProof master mix   100ul
    100uM Solexa_PCR_up      1ul
    100uM Solexa_PCR_lo      1ul
    H2O                   87.3ul
    50X SYBG I             0.8ul
    98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold
  File:2008-04-17-Solexa-lib-PCR.png

Perform another round of PCR on the 04/10/08 amplicon (27ng/ul)[edit]

   Template                    5ul
   10X Platinium Taq buffer   40ul
   50mM MgCl2                 12ul
   100uM AmpF6.2SolH         1.6ul
   100uM AmpR6.2SolH         1.6ul
   10mM dNTP                   8ul
   1mM dUTP                    2ul
   50X SYBG I                1.6ul
   H2O                       326ul
   JumpStart Taq(5U/ul)        4ul
   split into 4x100ul
   94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 6 -> 72C 3min -> 15C hold.
   Purified 4X100ul amplicons with 2x Zymo DNA concentrator-25 columns.
   Eluted DNA from each column with 30ul EB buffer. 
   Yield: 47ng/ul x 60ul.
   File:2008-04-12-Cpg-Jurkat-3rd-PCR-amplicons.png

Mme I digestion[edit]

  DNA                         30ul x 47ng/ul
  10X NEBuffer 4:              5ul
  1mM SAM                    2.5ul
  Mme I (2U/ul)                5ul
  H2O                         10ul
  37C 1h.
  Qiaquick purification -> 6% TBE PAGE size selection