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.
I estimated the concentration of each band based on the intensity of the 200bp band of the ladder.



Revision as of 23:38, 27 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).

  File:2008-03-27-colony-PCR.png

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 #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