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=== Preparing the LM-PCR === ==== QUICK RUN PCR to monitor cycle number ==== *The user guide has suggestions on the pcr condition for amplification of captured DNA *But since this is my first time doing the capture experiment, my capture efficiency might not be as high as expected. *Noi did the optimization before and I will modify her protocol. *I will do PCR -1 & +1 of the recommendation cycle number (16X). * I will use the amplification primers Noi used to amplify the libraries (TruS F/R). * I will use the same primer concentration as I used to amplify the library at 0.3uM instead of 0.5uM. * Noi had issue with evaporation when doing pcr in small volume, I will add 20ul mineral oil for the pcr reaction. {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" valign="bottom" | width="266" height="15" | Components | width="164" | Volume (ul) |- style="font-size:12pt" align="center" valign="bottom" | height="15" | KAPA HiFi HotStart ReadyMix (2X) | align="center" | 20.83 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | TruS F/R (10uM) > 0.3uM after adding template | align="center" | 1.25 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | H2O | align="center" | 2.92 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Total | align="center" | 25.00 |} # Aliquot 7.5ul MM + 5ul bead-bound captured DNA (total 12.5ul) 2X # Aliquot 7.5ul MM + 5ul ddH2O for NTC (total 12.5ul) 2X # Aliquot 4ul pcr section mix to pcr tubes # Ran PCR at 15,16 and 17 cycles on different thermocycler. '''LM-PCR program''' 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X, 17X --> 72C, 1min --> Hold @8C *TBE Gel [[File:ZhangLab_2 2015-01-21 15hr 31min.jpg|400px]] ==== Amplification in a large volume ==== '''Components''' '''Volume (ul)''' '''2.2rxn (ul)''' KAPA HiFi HotStart ReadyMix (2X) 50.00 110.00 TruS F/R (10uM) 3.00 6.60 H2O 7.00 15.40 '''Total''' '''60.00''' # Aliquot 63ul + 42ul bead-bound captured DNA (total ul) # Split 52ul 2X '''LM-PCR program''' 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 15X --> 72C, 1min --> Hold @8C :- Purified amplified captured DNA with Qiaquick PCR columns :- Elute with total volume 50ul of EB buffer, total 100ul for each set. :- Verify purified sequencing library in 6% TBE gel by loading 3ul. '''Library ID:''' 1 = HL-Seqcap-pNormalSetA-Jan15 2 = HL-Seqcap-pCancerSetA-Jan15 <br>
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