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==KAPA Library Quantification Kits== ==For Illumina Genome Analyzer Platform== ==Prepare Library Dilution Buffer== ==Prepare 10mM Tris HCL, pH 8.0+0.05% Tween 20== Need 1200ul for 1 sample *Make about 20ml *10ml*0.05%=5ul Tween20 *Mix 10ml 10mM Tris HCL with 5ul Tween 20 *Mix 6.4ml 31.25mM Tric-HCL with 13.6ml ddH2O and add in 10ul tween-20 ==Prepare qPCR Reagents== ===Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing=== *2X KAPA SYBR FAST qPCR Master Mix *6 DNA Standards *1:1000, 1:2000, 1:4000 & 1:8000 dilution of library DNA ==Prepare qPCR Master Mix== *Add 1ml of Illumina GA Primer Premix(10X) to 5ml bottle of KAPA SYBR FAST qPCR Master Mix (2X) and mix well ==Perform Serial Dilution of dsDNA Libraries== *Dilute purified library DNA in 10mM Tris HCL, pH 8.0+0.0.5% Tween 20 to 1:1000, 1:2000, 1:4000, 1:8000 dilution. 1:1000 {| {{table}} | Library Dilution Buffer||999ul |- | Library DNA||1ul |- | Total||1000ul |- | |} 1:2000 {| {{table}} | Library Dilution Buffer||100ul |- | Library DNA (1:1000)||100ul |- | Total||200ul |- | |} ==qPCR Setup== {| {{table}} | Setup||Volume(ul) |- | KAPA SYBR FAST qPCR Master Mix Containing Primer Premix||39.6 |- | PCR-grade water||13.2 |- | Diluted library DNA or DNA Standard (1-6)||13.2 |- | |} ==qPCR Cycling== Step1 95C 5M Step2 95C 30S Step3 60C 45S Step4 Plate Read Step5 Go to Step 2, Repeat 34X ==Analysis== *Annotate the DNA standards as follows before analyzing the data according to the qPCR instrument guidelines {| {{table}} | Sample Name||dsDNA Concentration (pM) |- | Std 1||20 |- | Std 2||2 |- | Std 3||0.2 |- | Std 4||0.02 |- | Std 5||0.002 |- | Std 6||0.0002 |- | |} ==Template== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- | align="center" width="65" height="12" align="center" valign="bottom" | | align="center" width="65" align="center" valign="bottom" | 1 | align="center" width="65" 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