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

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Check with Argarose gel, 4ul amplicons per lane, the ladder is Invitrogen Low Mass ladder (4ul).
Check with Argarose gel, 4ul amplicons per lane, the ladder is Invitrogen Low Mass ladder (4ul).
    [[Image:2008-03-27-colony-PCR.png|500px]]
I estimated the concentration of each band based on the intensity of the 200bp band of the ladder.
ExoSap-IT treatment: 5ul amplicons + 2ul ExoSap-IT, 37C 15min -> 80C 15min
    Samples submitted for sequencing
     Cpg #1:  Cpg A1        Cosmic #1: Cosmic A1
     Cpg #1:  Cpg A1        Cosmic #1: Cosmic A1
     Cpg #2:  Cpg B1        Cosmic #2: Cosmic B1
     Cpg #2:  Cpg B1        Cosmic #2: Cosmic B1
Line 77: Line 72:
     Cpg #8:  Cpg B2        E55kv1 #1: E55kv1 E1
     Cpg #8:  Cpg B2        E55kv1 #1: E55kv1 E1


===Analysis of sequencing results:
===Analysis of sequencing results===
               Insert(incl. H1&H2)  Target          Note
               Insert(incl. H1&H2)  Target          Note
   Cosmic #1:      235bp        FRMD4A exon  1 mismatch in H2
   Cosmic #1:      235bp        FRMD4A exon  1 mismatch in H2
Line 83: Line 78:
   Cosmic #3:      226bp        RIMS2 exon    100% match
   Cosmic #3:      226bp        RIMS2 exon    100% match
   Cosmic #4:    Similar to #2, also has amplification adaptors
   Cosmic #4:    Similar to #2, also has amplification adaptors
    
 
   Raw sequences of #2
  >Cosmic2-T7.ab1
  XXXXXXXXXXGCAGCTGAATAGGAACGATGAGCCTCCAACACCCAATCTT
  AAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCAGTCTACAACAGTGCT
  CTTCCAGTCTACCTAGCCTCATGCGTATCCGATCCAGAAGGCCAGAAATG
  GCAGTGTCGGACCTCGATAACATCTGCAGCTGAATAGGAACGATGAGCCT
  CCAACACCCAATCTTAAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCA
  GTCTACAACAGTGCTCTTCCAGTCTACCTAGCCTCATGCGTATCCGATCC
  AGAAGGCCAGAAATGGCAGTGTCGGACCTCGATAACATCTGCA 
====Cpg #1====
====Cpg #1====
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.
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.

Revision as of 22:43, 28 March 2008

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

                      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

   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)

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

Blunt end cloning

  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

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

             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   

Cpg #1

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