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

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===Circularization reaction===  
===Circularization reaction===  
==Exp.1 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==
==Probe design Version 2==
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.
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]]. I was able to design 505,592 probes covering 189,976 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 62,174,217bps. I selected a subset of probes covering chr1(25,554), chr22(12,683), chr21(6,780), and part of chr20(9,983). This is the second 55k chip synthesized at Agilent, called E55kv2 library. The adaptors and linker sequences follow the version 7 design.
                                  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.

Revision as of 23:54, 21 December 2007

Exp. 1 Perform another round of E55kv1 capturing experiment

Shearing template DNA

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

Exp.1 Exon capture experiment

   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

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. I was able to design 505,592 probes covering 189,976 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 62,174,217bps. I selected a subset of probes covering chr1(25,554), chr22(12,683), chr21(6,780), and part of chr20(9,983). This is the second 55k chip synthesized at Agilent, called E55kv2 library. The adaptors and linker sequences follow the version 7 design.