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=Low-input Bisulfite Conversion= * I will be working on the bisulfite conversion first protocol. ==Protocols Today== * Start with four tubes: 1571 cells, 314 cells, 157 cells, NTC, and cells that do not go through bisulfite conversion *Make 1571 cell/ul (original tube) *Make 314 cells/ul (dilute 1ul cells in 4 ul ddh2o) *Make 157cells/ul (dilute 1ul cells in 9 ul ddh2o) ===Cell Lysis=== * First, make protease Dilution **Add 3 uL Protease to 97 uL H2O for a 3:100 dilution **Add 1 uL Protease to 99 uL H2O for a 1:100 dilution * Next, add 1 uL 3:100 diluted Protease and 1 uL 3X Lysis Buffer (NP40) to 1 uL Frozen cells; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes * Cells are now lysed ===Bisulfite Conversion - Imprint Kit=== * Prepare DNA Modification Solution (CT Conversion Reagent): * Add 21 uL 0.5 mg/mL BSA and 1 uL Imprint Balance solution to each sample; incubate at 37 C for 10 minutes * Add 125 uL DNA Modification solution to each tube, incubate at 65 C for 90 minutes * Perform Column Purification of each sample: ** Elute into 8uL Elution Buffer ===MDA - Epitect Kit=== *Denature the no bisulfite conversion control by heating at 95C for 30 sec and cool down immediately with a cold block *Thaw REPLI-g Midi DNA Polymerase on ice. Thaw all other components at room temperature, vortex, then centrifuge briefly. *Add 1ul REPLI-g Midi DNA Polymerase to 29ul WBA reaction buffer *Mix and centrifuge briefly *Add 30ul Master mix to 10ul of bisulfite converted DNA *Incubate at 28C for 1 h *inactivate at 65C for 10 min ===MinElute === *Elute with 10ul buffer *Store at -20C ===Bisulfite Conversion Efficiency Test=== ===Nanodrop=== ===Tagmentation - Shendure Protocol=== *Take 3ul of the MDA product for 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 * Next, add 1 uL 1:100 diluted Protease to solution; incubate at 37 C for 30 minutes, followed by 70 C for 20 minutes ===Amplification - Kapa + BST=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume''' | align="center" style="background:#f0f0f0;"|'''MM''' |- | Nuclease Free H2O||16||140.8 |- | 10uM Orange Primer||1||8.8 |- | 10uM Blue Primer||1||8.8 |- | Tagmented DNA||6||Do not add to MM |- | 2X Kapa sybr supermix||25||220 |- | BST||1||8.8 |- | Total||50||387.2 |} *Transfer DNA to strip tube and add 44ul MM to each reaction * Raise volume of any non-bisulfite converted samples to 10 uL (add 2 uL H2O) * Add 1 uL Orange Primer, 1 uL Blue Primer, 25 uL KAPA SYBR Supermix, 1 uL BST Polymerase or Klenow Polymerase, and 12 uL H2O to each sample * Perform qPCR using the following reaction conditions: ** 60 C, 20 min incubation (BST second strand synthesis) ** 72 C, 3 min incubation (Nextera extension (where necessary; not needed if using Klenow)) ** 95 C, 30 seconds (Denature) ** 35 cycles of: *** 95 C, 10 seconds (Denature) *** 58 C, 30 seconds (Anneal) *** 72 C, 3 minutes (Extend) ** 72 C, 5 minutes (final extension) ** Hold at 4 C [[File:08012011.jpg|1000px]] *Perform qiaquick column, will decide wether we want to do cloning or not ===PAGE Gel Analysis=== [[File:ZhangLab_2 2011-08-01 16hr 08min.jpg|400px]] ===Results=== *Results are kind of surprising, 300 cells library are amplified before 1500 cells for the bisulfite converted samples. Could it be contamination? *For non bisulfite converted 150 and 300 cells' library there are almost no difference in the Ct value. We had a similar results from the experiment done on the July 28th, were we didn't see much difference in theCt value. This could be due to performance issue with the epitect kit on non-bisulfite converted samples. *We are not sure if the efficiency of the epitect kit on regular gDNA, repeating the experiment with our own MDA reaction mix will probably draw a better conclusion for we know that the Phi29+N9 primer do work on regular gDNA
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