Kun:LabNotes/ExonomeSeq: Difference between revisions
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*Two Agilent probe designs that allow for long linkers were tested: | *Two Agilent probe designs that allow for long linkers were tested: | ||
**[[Media:Amplifiable_MIP_design_KZ_v10_DpnII.docx|TypeIIs design:]][[Media:primers2padlock-TypeIIs.txt|[Codes]]] this is similar to the previous designs in that AP1V4/AP2V4 sequences that contain TspRI/DpnII Type IIs cutting sites were used. | **[[Media:Amplifiable_MIP_design_KZ_v10_DpnII.docx|TypeIIs design:]][[Media:primers2padlock-TypeIIs.txt|[Codes]]] this is similar to the previous designs in that AP1V4/AP2V4 sequences that contain TspRI/DpnII Type IIs cutting sites were used. | ||
**[[Media: | **[[Media:Amplifiable_MIP_design_KZ_v10_NickingE.docx|Nicking Enzymes:]][[Media:primers2padlock-NickingE.txt|[Codes]]] in this design, AP1V4/AP2V4 were replaced with eMIP_CA1_F/eMIP_CA1_R used by Jay Shendure in his eMIP 55k probe design. eMIP_CA1_F/eMIP_CA1_R contain recognition sites for nicking enzymes Nt.AlwI and Nb.BsrDl. Using nicking enzymes will simplified the probe prep step, also the digestion could be more complete and hence the yield could be higher (yet to be validated). The reason I stayed away from the method previously is that a small fraction of probes that contain the cutting sites within H1/H2 are not compatible with this method. My current thinking is that if it does turn out to be simpler and more efficient, we can make the majority of probe using this method, and the incompatible probes with the DpnII/USER method. | ||
===The first probe set: [[Kun:LabNotes/ExonomeSeq/2007-11-15]]=== | ===The first probe set: [[Kun:LabNotes/ExonomeSeq/2007-11-15]]=== |
Revision as of 07:29, 26 October 2008
2008 <calendar> name=Kun:LabNotes/ExonomeSeq format=%name/%year-%month-%day date=2008/02/01 view=oneyear </calendar>
2007 <calendar> name=Kun:LabNotes/ExonomeSeq format=%name/%year-%month-%day date=2007/11/01 view=threemonths </calendar>
Probe Design
Oct08 whole exome probe set
- Athurva created a list of non-redundant coding exons for all RefSeq genes.
- Athurva has converted my Cpg probe design program for exon probe design. He has made a number of changes including the strategy to search for a optimal set of probes for a large exon. I made additional changes on the codes. Here is the current version of probe design program.
- A 27k probe set was ordered on 10-25-2008. This probe set contains four subset of oligos for different designs/projects.
- Mike Chou ChrX probes(Len >=100bp, TypeIIs): 5904
- Kun Chr21/22 probes (Nicking Enzymes): 2924+5654
- Kun Chr20 probes (TypeIIs): 7334
- MCC_probes(Celso/Feng, Bing Ren's lab): 5184
- Two Agilent probe designs that allow for long linkers were tested:
- TypeIIs design:[Codes] this is similar to the previous designs in that AP1V4/AP2V4 sequences that contain TspRI/DpnII Type IIs cutting sites were used.
- Nicking Enzymes:[Codes] in this design, AP1V4/AP2V4 were replaced with eMIP_CA1_F/eMIP_CA1_R used by Jay Shendure in his eMIP 55k probe design. eMIP_CA1_F/eMIP_CA1_R contain recognition sites for nicking enzymes Nt.AlwI and Nb.BsrDl. Using nicking enzymes will simplified the probe prep step, also the digestion could be more complete and hence the yield could be higher (yet to be validated). The reason I stayed away from the method previously is that a small fraction of probes that contain the cutting sites within H1/H2 are not compatible with this method. My current thinking is that if it does turn out to be simpler and more efficient, we can make the majority of probe using this method, and the incompatible probes with the DpnII/USER method.
The first probe set: Kun:LabNotes/ExonomeSeq/2007-11-15
- One 55k library received on 12/12/2007, OLID: 018274110001, 10pmole, resuspended in 500ul RT-PCR grade water (new vial) -> 20nM x 500ul -> Take 100ul out to a 1.5ml non-stick tube as the working tube.
- The second probe set: Kun:LabNotes/ExonomeSeq/2007-12-21
- Two Agilent 55k libraries were ordered on 01/04/2008.
- Cosmic3760 0189191
- CpgCes: Cpg28k + CES27k 0189181
Received 01/24/2008. 10pmole, resuspended in 500ul RT-PCR grade water (new vial) -> 20nM x 500ul -> Take 100ul out to a 1.5ml non-stick tube as the working tube.
Overview of experiments
Sequencing library construction
Random primed
- Forward primer (SolFN6):CGACGCTCTTCCGATCTNNNNNN Tm left = 59.20C
- Reverse primer (SolRN6):CATACGAGCTCTTCCGATCTNNNNNN Tm left = 59.45C