tag:blogger.com,1999:blog-6334453475526523597.post1073208879538276649..comments2017-07-04T13:19:44.773+01:00Comments on CoreGenomics: Agilent tools to help with your NGS poolingJames@cancerhttp://www.blogger.com/profile/02825715598810395734noreply@blogger.comBlogger7125tag:blogger.com,1999:blog-6334453475526523597.post-86040321606551937872017-05-04T15:29:10.679+01:002017-05-04T15:29:10.679+01:00Hello,
I had a question regarding the Post...Hello,<br /><br /> I had a question regarding the Post capture index tagged Library concentration for SureSelect XT? .Does it have to be within the range of 10nm-25nm? The example above shows C(i) within that range.<br /><br />Is it ok if concentration of Index Tagged library is High (70-100nm). Can we dilute and Pool it to 10nm. Will it affect capture performance and sequencing?<br /><br />Thanks!Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-58700899544224337072016-07-29T10:47:36.247+01:002016-07-29T10:47:36.247+01:00Stick the formula into Excel if you prefer. Your l...Stick the formula into Excel if you prefer. Your libraries are almost equivalent so there is no need to use this formula on this occasion. Simply mix them at 3ul each and add 13.8 of low TE to get approximately a 10nM stock. We'd always check this stock with qPCR before clustering.James@cancerhttps://www.blogger.com/profile/02825715598810395734noreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-36167652692066075122016-07-27T02:14:20.461+01:002016-07-27T02:14:20.461+01:00I had a few questions to ask about the pooling of ...I had a few questions to ask about the pooling of libraries for agilent sureselect assay. I working with 3 samples, based on my bioanalyzer results ,my Index tagged DNA Concentration is (sample1): 76nM, (sample2):76nM and (sample2): 79nM. <br /><br />So, with this formula above for sample 1<br /><br /> C(i)=76<br />v (f)=20<br />#=3<br />c(F)=10<br /><br />Volume to use (sample 1) =0.87<br /><br /><br />Volume to use (sample 2)=0.87<br /><br /><br />Volume to use (sample 3)=0.84<br /><br />Please can anyone tell me if this is correct. I will be sequencing it on Nextseq 500(Midoutput kit)150 cycles with a seeding concentration of 1.3pM<br />Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-40138060490772272852016-04-25T22:36:18.543+01:002016-04-25T22:36:18.543+01:00I'm using an excel sheet for calculate this.I'm using an excel sheet for calculate this.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-45396843924016680332016-04-25T22:34:11.359+01:002016-04-25T22:34:11.359+01:00I can not understand how can you set the final des...I can not understand how can you set the final desired volume of the pool and use this value for each library, since the final volume will depend on the other libraries in the pool. Of course, if you end up with less than the desired volume, TE can be used, but using this method, your total volume will probably be higher than the desired.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-58652043173311316682014-06-21T12:24:48.649+01:002014-06-21T12:24:48.649+01:00Large variations might also suggest that the start...Large variations might also suggest that the starting samples were not the same quality or quantity as each other or something happened during library-prep. This itself can be a useful QC.James@cancerhttps://www.blogger.com/profile/02825715598810395734noreply@blogger.comtag:blogger.com,1999:blog-6334453475526523597.post-4623713175449564692014-06-19T15:34:05.042+01:002014-06-19T15:34:05.042+01:00I've used this method before. One massive adva...I've used this method before. One massive advantage is that you can normalise up if you have a few low concentration samples (as long as you have sufficient higher concentration samples to balance the total volume). Just add a larger amount for the those low samples and remove any excess volume from the amount of low TE buffer you need to add.<br />The downside to this method is you need to start with samples roughly the same (as in the example above). Large variations in starting concentrations leads to having to pipette either large or small volumes.Unknownhttps://www.blogger.com/profile/14869462307678967101noreply@blogger.com