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==Protocol== #SplintR reaction for Samples 1-4 #*Incubate at 25C for 20min #*Heat inactivate at 65C for 20min {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Sample | width="51" | 10X SplintR Buffer | width="51" | SplintR Ligase | width="51" | 10uM ppBCL11B | width="51" | 10uM ppCUX2 | width="51" | 10uM ppGFAP | width="51" | 10uM ppRELN_1 | width="51" | gDNA 12878 80.3ng/ul | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | align="right" | 3 | align="right" | 3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 3 | align="right" | 19.8 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | align="right" | 3 | align="right" | 3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0 | align="right" | 22.8 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | align="right" | 3 | align="right" | 3 | align="right" | 0.3 | align="right" | 0 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0 | align="right" | 23.1 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | align="right" | 0 | align="right" | 0 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0.3 | align="right" | 0 | align="right" | 28.8 | align="right" | 30 |} #Circligase II reaction with 400nM total #*Incubate at 60C for 1hr #*Inactive at 80C for 10min {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Components | width="51" | Volume |- style="font-size:11pt" valign="bottom" | height="14" | CircLigase II 10X Buffer | align="right" | 3 |- style="font-size:11pt" valign="bottom" | height="14" | CircLigase II Enzyme | align="right" | 1.5 |- style="font-size:11pt" valign="bottom" | height="14" | 5M Betaine | align="right" | 6 |- style="font-size:11pt" valign="bottom" | height="14" | 50mM MnCl2 | align="right" | 1.5 |- style="font-size:11pt" valign="bottom" | height="14" | 10uM ppBCL11B | align="right" | 0.3 |- style="font-size:11pt" valign="bottom" | height="14" | 10uM ppCUX2 | align="right" | 0.3 |- style="font-size:11pt" valign="bottom" | height="14" | 10uM ppGFAP | align="right" | 0.3 |- style="font-size:11pt" valign="bottom" | height="14" | 10uM ppRELN_1 | align="right" | 0.3 |- style="font-size:11pt" valign="bottom" | height="14" | H2O | align="right" | 16.8 |- style="font-size:11pt" valign="bottom" | height="14" | Total | align="right" | 30 |} #Split each sample into 2 15ul samples #Exo I/III digest one of each sample by adding 1ul Exo I and 1ul Exo III #*Incubate at 37C for 1.5 hour #Purify all 10 samples with Zymo ssDNA/RNA columns #*Elute 15ul each #qPCR all 10 samples {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''16X Volume''' |- | Captured template||5||0 |- | 10uM ISB_CA_AF||0.4||6.4 |- | 10uM ISB_CA_AR.T1||0.4||6.4 |- | 2X KAPA SYBG MM||12.5||200 |- | H2O||6.7||107.2 |- | Total||25||320 |} *Aliquot 20ul from 16X master mix and add 5ul captured template Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min [[File:20170408_SplintRNoTemplateTest_qPCR.PNG|450px]] #Run gel on both ##PP + gDNA + SplintR ##PP + SplintR ##PP -ppCUX2 + SplintR ##PP (negative control for qPCR) ##PP + CircLigase II (positive control) ##PP + gDNA + SplintR (digested) ##PP + SplintR (digested) ##PP -ppCUX2 + SplintR (digested) ##PP (negative control for qPCR) (digested) ##PP + CircLigase II (positive control) (digested) [[File:2017-04-08_SplintR_DNAsplint_Test.jpg|450px]]
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