ࡱ> 241c bjbj** .HSbHSb VRR8.AjJJ(rrr$a d-rr4rrrǪ5@^0A 0  A RB : KAPA library quantification Designed for easy library quantification, these reactions seem annoyingly delicate and prone to fail for no apparent reason. The following protocol seems to be robust if followed precisely. Hence the pedantic level of detail. Use KAPA Library Quantification Kit for Illumina. These kits are very expensive use them sensibly (ie: wait until you have 10 or more libraries to test otherwise we run out of standards very quickly). Use non-filtered tips throughout except for handling your libraries and the stock reagents. Do NOT use siliconized tips or tubes. We use Bio Rad CFX-96 machines with recommended Bio Rad plates/seals. Previous versions of this protocol have noted that you should avoid the edge wells on a 96 well plate. Having moved to skirted StarLabs plates, this is no longer an issue and the whole plate can be used. Dilute libraries 0.5l in 100l Library Dilution Buffer in 1.5ml tubes, vortex Dilute again 2l in 100l Library Dilution Buffer in 1.5ml tubes, vortex (final dilution is 1:10,000) Defrost KAPA standards and KAPA mix (add oligos to KAPA mix if not already added) Make a master mix of 6l KAPA mix and 2l water per reaction, add 5 more reactions as this solution does not pipette very accurately. Ie: for 24 libraries, make enough for 65 reactions, which is 390l KAPA mix and 130l water Set up qPCR reactions in 96 well plate: Each reaction contains 8l master mix 2l DNA Perform 2 replicates for each standard and 2 replicates for each 1:10,000 library dilution PCR program: 95 5min 95 30s \ 60 45s x35 +acquire / Library Dilution Buffer: 10mM Tris pH8.0 0.05% Tween 20 Analysis Use Excel to plot the cycle number for each standard against the concentration (20, 2, 0.2, 0.02, 0.002, 0.0002 pM). They should form a straight line when the concentration is on a log scale. Use the trend line option (exponential) to get a formula for the line, then check this by calculating the measured concentrations of the standards based on cycle number they should be very close to the actual concentrations. Use the same formula to calculate the concentration of each library, and correct for the 1:10,000 dilution. Correct for the average fragment size in your library (get this from the Bioanalyzer profile) corrected conc = measured conc from KAPA x 452 / average fragment size The concentrations obtained by Bioanalyzer and KAPA should be within 2-fold, remember that Bioanalyzer sample was diluted before running.      FILENAME KAPA library quantification v1.3 Houseley lab  PAGE 2 } a b c w x   J V m &(::DŽŽŽرررررŽŭحɡɡɡhXOJQJh5*eh>-<OJQJh)h({OJQJh({hXh h4Vh4Vh< h4V6h4VhMh)5hM5CJ(aJ(h({5CJ(aJ(? b c   l m 8: p^p`gd({gd< 0^`0gd< 0^`0gd4Vgd)$a$gd) BUV`a"q x`gdmxgdmgd>-1<Jȸȴjh(0JU*h0JmHnHu* h(0Jjh(0JUh%h(hhYhnmHnHu hXh(jh(UhLjhLU h)h5*eh{|h5*eh5*e5 h<5h5*ehXh({%xgdm,1h. A!"#$% s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberH"H n Balloon TextCJOJQJ^JaJN/1N nBalloon Text CharCJOJQJ^JaJPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< +  HHUUUX   3IPRX!@ @H 0(  0(  B S  ?egCI   ! ) , m ) , 30bcc} JVjjmPo}I I     ' , 0bcc} JVjjmPo}I I , 0/XMmp.%*Mr2s7<; BcH7TYs,[bQycRd5*egxomwnuLu({./|fL0{|(>-s7LbR4Vnn()Ymd @(33+ @ @UnknownG.[x Times New Roman5Symbol3. .[x Arial7..{$ Calibri9. . Segoe UIA$BCambria Math"1h\HG녧tHG |w]w]!4 2qHP ?72!xxBA  Prepare 0jhousele Jon Houseley Oh+'0   @ L X dpx Prepare 0 jhouseleNormalJon Houseley12Microsoft Office Word@R@|s@(.@5w] ՜.+,0 hp  University of Edinburgh   Prepare 0 Title  "#$%&'(*+,-./03Root Entry FǪ551Table WordDocument.SummaryInformation(!DocumentSummaryInformation8)CompObjr  F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q