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==Bisulfite Conversion DNA Damage Estimation== *Perform PCR with CHR8, CHR21 and CHR22 * These will be our standards [[File:ZhangLab_2 2011-05-10 10hr 40min.jpg|500px]] * Need to purify CHR8, CHR21, and CHR22. **Qubit Results(ng/ul) **Assume amplicon length ~200bp *CHR8-3.25ng/ul 26.72nM *CHR21-4.39ng/ul 36.09nM *CHR22-4.77ng/ul 39.21nM *It is better to perform a size selection, since there are some ghosting on the gel ===Size Selection=== [[File:ZhangLab_2 2011-05-26 17hr 09min.jpg|400px]] *Prepare 3 gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel. *Cut out the correct band (~200 nt in this case), transfer gel slice to shearing assemblies *Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. *Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. *Spin down all tubes at 13,000rpm for 2min *In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube. *Perform ethanol precipitation *Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Needed''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- |100% Ethanol||align="right"|2.5X Sample Volume||align="right"|950uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|38uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|2uL |- |Sample||align="right"|Sample Volume||align="right"|380uL |- |Total Volume |colspan="2" align="right"|1370uL |- |} *Mix all content by repeat pipetting using multichannel pipette *Store in -80C for more than 20 mins or forever *Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge *Centrifuge frozen 1.5mL tubes at 10000rpm for 30m at 4C *Remove all liquid '''CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE''' *Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube *Centrifuge at 10,000rpm for 5min at 4C *Remove all liquid, air dry sample for 5min or until samples dried out *Resuspend the DNA with 20uL RNAse free H2O ===Qubit Quantifiction=== *CHR8-1.87ng/ul 15.37nM *CHR21-0.545ng/ul 4.48nM *CHR22-1.12ng/ul 9.21nM ===Determine the efficiency of bisulfite conversion=== *After performing the bisulfite conversion procedures, we should perform real-time PCR of the samples and controls to determine the amount of converted human genomic DNA. Base on the we can determine the conversion efficiency. ===Make Serial Dilution on Control DNA=== *number of copies = ( amount * 6.022x1023) / (length * 1x10^9 * 650) *Calculation for STD based on 200bp and 8ul of the dsDNA *Create 4 STDs *1:1000 dilution from 20pM to 0.02pM *1:1000 dilution from 0.02pm to 0.00002pM (this is STD1) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample Name''' | align="center" style="background:#f0f0f0;"|'''Concentration (pM)''' | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | align="center" style="background:#f0f0f0;"|'''DNA Molecule''' |- | STD1||0.00002||1.94E-05||90100 |- | STD2||0.000002||1.94E-06||9010 |- | STD3||0.0000002||1.94E-07||901 |- | STD4||0.00000002||1.94E-08||90.1 |} *With starting amount of 600pg, assume the % lost of sample is 90, 10 and 1. The # of molecules of resulting DNA will be as follows. {| {{table}} | align="center" style="background:#f0f0f0;"|'''%''' | align="center" style="background:#f0f0f0;"|'''ng''' | align="center" style="background:#f0f0f0;"|'''DNA Molecule''' |- | 100||0.6||86.9 |- | 90||0.54||78.2 |- | 10||0.06||8.69 |- | 1||0.006||0.869 |} ==Prepare qPCR Reagents== ===Ensure that the following reagents are completely thawed and thoroughly mixed by vortexing=== *2X KAPA SYBR FAST qPCR Master Mix *6 DNA Standards *1:1000 dilution of bisulfite converted library DNA *Primer Mix (F+R) 1:1 ratio 1.65uM ==Prepare qPCR== {| {{table}} | ||CHR8||CHR21||CHR22 |- | 2X iQ Super Mix||20||20||20 |- | Primer F+R (1.65uM)||4.85||4.85||4.85 |- | gDNA||8||8||8 |- | ddH2O||7.15||7.15||7.15 |- | Total||40||40||40 |} ==Perform real-time PCR reaction in thermocycler== 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 ==Generate Standard Curve== ===For Bio-Rad Real time Thermal Cycler=== *Edit plate setup *Select and add wells with control DNA as Standard *Specify Quant Standards (bottom right button) *After PCR cycle *Select Quantitation Tab on left column to see standard curve *Select Calculations Tab at the bottom to see the sample concentrations ==For Eppedorf Real time Thermal Cycler== *Edit Plate Layout *Select wells for standards *right click on well and select well as standard *Input name and standard amount *After PCR cycle *Standard curve will show up on the bottom half of the analysis screen *Sample concentrations will show up on left column ==Generate Recovery Curve==
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