Kun:LabNotes/ExonomeSeq/2007-12-21: Difference between revisions

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===Circularization reaction===  
===Circularization reaction===  
==Exp.2 Exon capture experiment==
     Sheared NA10835(~300ng/ul)        3ul
     Sheared NA10835(~300ng/ul)        3ul
     E55kv1 probes 107nM              17ul
     E55kv1 probes 107nM              17ul
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95C 10min -> 60C 40h -> add 2.5ul NSL mix -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold
95C 10min -> 60C 40h -> add 2.5ul NSL mix -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold
==Colony PCR==
Yesterday I pooled the transformations for 180/280bp and 470bp fragment in one plate, and then 150bp and 350bp fragments in another plate. I have only <10 colonies in the first plate and >200 colonies in the second. Today I decided to pick 8 colonies from each plate for PCR.
                                  X 17
      2X Taq master mix:  15ul    255ul
      10uM M13 F/R mix:    1ul    17ul
      H2O                  14ul    238ul


94C 3min -> (94C 30sec -> 55C 30sec -> 72C 1min) x 35 -> 72C 3min -> 4C hold.
==Probe design Version 2==
I came up with an iterative primer searching program when working on the design of CpG island probes. I decided to modify this program for exonome probe design since  it will generate probes for many regions that were missing by Keith's program. Here is the [[Media:getExonicPrimers.txt|code of my program]]. The gap for fill-ins is 175-225bp.  I was able to design 381,765 probes covering 183,448 exons in the human RefSeq genes. The total length of exonic sequences captured is 80,719,171bps. The total non-overlapping genomic regions covered by this probe set is 68,730,151bps. I selected a subset of 55,067 probes targeting the first 3760 genes in the Cosmic gene database. The last 67 probes were removed to make a 55k set. This is the second 55k exonic chip synthesized at Agilent, called Cosmic3760 library. I used the V4 adaptors and the V7 linker. The reason of using V4 adaptor is to have the option of using BciVI.
*[[Media:ECosmic3760_probes.txt.gz|Probe information file.]]
*[[Media:Cosmic3760.seq.gz|Raw sequences of all 55,000 probes.]]

Latest revision as of 00:18, 26 January 2008

Exp. 1 Perform another round of E55kv1 capturing experiment[edit]

Shearing template DNA[edit]

10ul 353ng/ul GM10835 gDNA + 750ul shearing buffer (1X TE w/ 10% Glycerol) -> sheared with Nebulizer with unknown pressure for 1min -> recovered 650ul -> isopropanol precipitation with the presence of 4ul linear acrylamide.

Resuspended in 10ul ddH2O -> ~300ng/ul

Circularization reaction[edit]

Exp.2 Exon capture experiment[edit]

   Sheared NA10835(~300ng/ul)        3ul
   E55kv1 probes 107nM              17ul
   10X Ampligase buffer           2.25ul

95C 10min -> 60C 40h -> add 2.5ul NSL mix -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold

Probe design Version 2[edit]

I came up with an iterative primer searching program when working on the design of CpG island probes. I decided to modify this program for exonome probe design since it will generate probes for many regions that were missing by Keith's program. Here is the code of my program. The gap for fill-ins is 175-225bp. I was able to design 381,765 probes covering 183,448 exons in the human RefSeq genes. The total length of exonic sequences captured is 80,719,171bps. The total non-overlapping genomic regions covered by this probe set is 68,730,151bps. I selected a subset of 55,067 probes targeting the first 3760 genes in the Cosmic gene database. The last 67 probes were removed to make a 55k set. This is the second 55k exonic chip synthesized at Agilent, called Cosmic3760 library. I used the V4 adaptors and the V7 linker. The reason of using V4 adaptor is to have the option of using BciVI.