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==Continue on 8/28== *Denature all samples at 95C for 30sec and quickly transfer to cooler rack {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | 10X Repli Phi Buffer||2||8.8 |- | 1mM N9 Primer||1||4.4 |- | 25mM dNTP||0.8||3.52 |- | Repliphi Phi 29 (100U/ul)||1||4.4 |- | Klenow Exo Minus (10U/ul)||0.5||2.2 |- | Nuclease free H20||4.7||20.68 |- | Modified DNA Sample||10||Do not add to MM |- | Total||20||44 |} *Add 10ul of MDA mastermix to each sample *30C 60 min, 65C 10 min, 4C hold ===MinElute === *Elute with 10ul nuclease free water ===Speedvac=== *To get 3ul for shendure's method of nextera library tagmentation, I will transfer the 10ul samples to qpcr tube, put the cap on and puncture a hole at the cap and speedvac till the sample is dry *took about 28min to dry samples *Re-dissolve the samples with 3ul of nuclease free water, sit at RT for 5 min ===Tagmentation=== * To lysate, add 1 uL 1:50 diluted Nextera Transposase and 1 uL 5x Nextera HMW Buffer; incubate at 55 C for 5 minutes *To make 1:100 Protease, mix 1ul of protease with 99ul of nuclease free water * Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes *Prepare mastermix for library amplification {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | Nuclease Free H2O||16||70.4 |- | 10uM Orange Primer||1||4.4 |- | 10uM Blue Primer||1||4.4 |- | Tagmented DNA||6||Do not add to MM |- | 2X Kapa SYBR Supermix||25||110 |- | BST||1||4.4 |- | Total||50||193.6 |} *Add 44ul MM to each reaction * Perform qPCR using the following reaction conditions: ** 65 C, 3 min incubation (BST gap filling) ** 95 C, 30 seconds (Denature) ** 35 cycles of: *** 95 C, 10 seconds (Denature) *** 58 C, 30 seconds (Anneal) *** 72 C, 3 minutes (Extension) ** 72 C, 5 minutes (final extension) ** Hold at 4 C *300 cells was taken out after 7 cycles *150 cells & NTC was taken out after 12 cycles [[File:ZhangLab_2 2011-08-28.jpg|700px]] ===Purification=== *Minelute column, elute with 15ul water *Nanodrop gave me weird results, I was having problem when measuring the blank sterile water. Will avoid using the water provided by the Repliphi phi 29 kit for elution next time **150:6.6ng/ul **300:-0.2ng/ul *Use Qubit to measure the concentration instead **150:4.16ng/ul **300:1.46ng/ul *Looks like I do not have enough samples for end repair, may be I should run a few more cycles of PCR? * I would like to run a gel to confirm the size of the amplicons
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