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==MDA== *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||6.2 |- | 1mM N9 Buffer||1||3.1 |- | 25mM dNTP||0.8||2.48 |- | Repliphi Phi 29 (100U/ul)||1||3.1 |- | Klenow Exo Minus (10U/ul)||0.5||1.55 |- | Nuclease free H2O||4.7||14.57 |- | Modified DNA Sample||10||Do not add to MM |- | Total||20||31 |} *Add 10ul of MDA mastermix to each sample *30C 60 min, 65C 10 min, 4C hold ===EtOH Precipitation=== *Elute with 3ul nuclease free water *Incubate at RT for at least 10min *Transfer samples to qPCR strip tubes for tagmentation and qPCR ===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) ** 20 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 *Stopped reaction at cycle 17 *Purify with minelute, elute with 15ul of EB buffer, keep 1ul for gel ===Bisulfite Verification w CHR8,21&22=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''MM''' | align="center" style="background:#f0f0f0;"|'''CHR8''' | align="center" style="background:#f0f0f0;"|'''CHR21''' | align="center" style="background:#f0f0f0;"|'''CHR22''' |- | 2X iQ Super Mix||20||20||20 |- | Primer F (3.3uM)||6||6||6 |- | Primer R (3.3uM)||6||6||6 |- | gDNA||2||2||2 |- | H2O||6||6||6 |- | Total||40||40||40 |} Step1 96C, 3m Step2 95C, 30s Step3 62C, 1m Step4 72C, 1m Step5 Go to step2 repeat 39 times Step6 72C, 5m Step7 4C, Forever ===Adding Purple and Pink Nextera Adapters=== *Using barcode 8 here {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Per Reaction''' |- | DNA||Do not add to MM |- | Kapa HF Mix||55 |- | Adaptor 1||2.2 |- | Adapor 2 (Barcode)||2.2 |- | H2O||17.6 |- | Total||77 |} *Added 0.4ul 50X SYBR Green to each reaction Nextera_2nd 95C 30sec [95C 10sec; 60C 30sec; 72C 2min] X 2 [95C 10sec; 62C 30sec; 72C 2min] X 3 72C, 3min ===Page Gel Analysis=== *I will run a gel with the CHR8,21,22 samples and the blue/orange primers amplified samples w. -ve control [[File:ZhangLab_2 2011-10-06 19hr 09min.jpg|thumb|400px]] ===Results=== *The bisulfite conversion pcr test failed for some reason. Mostly due to old sodium bisulfite reagent. It was stored at -20C as recommended, but it is better to work with freshly prepared bisulfite conversion reagents. Will spend more time to investigate on this if possible. *The 1st amplification worked out really nice, with the exact amplicon size as expected and negative control didn't get amplified. *However after the 2nd amplification, unspecific bands showed up for both the sample and the control. I suspect this is due to lost of samples due to purification using the column. *I would like to try Rui's protocol where she did purification with beads and she had pretty good results from it.
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