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='''EZ DNA Methylation Direct-GM20431 PCR analysis '''= ==Objective== *Confirmation of the bisulfite conversion of GM20431 for 80,000 cells ==Samples & Materials== *GM20431 *EZ DNA Methylation Direct Kit *Primers - From IDT -------------------------------------------------- 0.1_F_chr22_31384238GTGAATAGGTTAAGTGAGGTAGAAG 0.1_R_chr22_31384238AAAAAAATCAAACACCAACTATAAA 0.8_F_chr21_39672131AAAATATTGGGATTATAGGTATGAGT 0.8_R_chr21_39672131AACTTCTAAACTAACCAAAACAAAA 0.9_F_chr8_119031762TTATAGTTTGGGTGATAGAGTAAGATT 0.9_R_chr8_119031762AAACCCTAAACAAAATACTCAATATAA -------------------------------------------------- *Creating 100uM primer 0.1_F_chr22 27.9nmol RNAse free H2O 279uL 0.1_R_chr22 31.8nmol RNAse free H2O 318uL 0.8_F_chr21 31.30nmol RNAse free H2O 313uL 0.8_R_chr21 31.70nmol RNAse free H2O 317uL 0.9_F_chr8 30.10nmol RNAse free H2O 301uL 0.9_R_chr8 29.30nmol RNAse free H2O 293uL ------------------------------ *Making 3.3uM primer working solution Dilute 33uL 100uM primer with 967uL RNAse free H2O *Agarose Gel ==Overview== *Cell Preparation *Bisulfite Conversion *PCR amplification *Agarose Gel Electrophoresis ==Procedures== *Cell Preparation Resuspended cells in T25 flask by repeat pipetting Perform cell counting aspirate 15mL cell suspension and transfer to a 50mL centrifuge tube Spin down at 10,000g for 3 mins Aspirate supernatant completely Resuspended cells with 622uL UV treated PBS *Cell Counting: 366,367cells/mL * 15mL = 4,978,231.6 cells Resuspend cells to 8000cells/uL 4,978,231.6cells/(8000cells/uL)=622uL *Cell Dilution Performed cell dilution to reach concentration of 8000cells/uL 800cells/uL 500cells/uL 100cells/uL 5000cells/uL 1000cells/uL *Sample Digestion with Proteinase K 1rxn x 6 ------------------------------------ RNAse-free H2O 2.0 Protinase K 1.0 M-Digestion Buffer (2X) 13.0 ------------------------------- Mix by repeat pipetting Cells 10.0 ------------------------------------ 26uL Incubate the samples at 50C for 20 mins Mix content by vortexting and centrifuge for 5 min at 10,000g *Bisulfite Conversion of DNA Add in 130uL of CT conversion Reagent Solution into a labeled PCR tube Extract 20uL of sample supernatant Mix by repeat pipetting Centrifuge briefly to ensure no droplets are in the cap or side of the tube Perform reaction in thermocycler Step1 98C, 8m Step2 64C, 3.5hr Step3 62C, 1m Step4 4C, storage for up to 20 hr Add 600uL of M-Bindin Buffer into a IC Column and place column into a collection tube Load samples into IC column Close cap and mix by inverting the column several times Centrifuge at (max speed) 20,000g for 30s Discard flow through Add 100uL M-Wash Buffer to column Repeat Centrifuge Add 200uL of M-Desulphonation Buffer to column let stand at RT for 20m Repeat centrifuge step Add 200uL of M-Wash Buffer to the column Repeat Centrifuge Repeat washing step Place column in a 1.5mL tube Add in 10uL of M-Elution Buffer directly to the column matix Repeat Centrifuge *DNA concentration measurement Dilute sample 1/10 concentration: 12.3ng/uL -0.5uL of stock sample should be enough for PCR *PCR A B C D E ------------------------------ Taq 2X Master Mix 20 20 20 20 20 Primer F (3.3uM) 6 6 6 2 2 Primer R (3.3uM) 6 6 6 gDNA 0.5 0.5 0.5 0.5 0 RNAse free H20 7.5 7.5 7.5 15.5 16 ------------------------------- 40uL 38uL 38uL A-CHR22 F/R B-CHR21 F/R C-CHR8 F/R D-H18S F/R E-H18S F/R Perform 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 *1st Gel Electrophoresis Well 1 2 3 4 5 6 7 8 ------------------------------------------------------------- Content Blank CHR22 CHR21 CHR8 H18S H18S Ladder Sample 0 3 3 3 3 3 1 0 0.5X TBE Buffer 0 3 3 3 3 3 3 0 6X Loading Dye 0 3 3 3 3 3 3 0 ------------------------------------------------------------- **Run at 135 V for 20 min. *2nd Gel Electrophoresis Well 1 2 3 4 5 6 7 8 ------------------------------------------------------------- Content Blank CHR22 CHR21 CHR8 H18S H18S Ladder Sample 0 6 6 6 6 6 1 0 0.5X TBE Buffer 0 4 4 4 4 4 4 0 6X Loading Dye 0 4 4 4 4 4 4 0 ------------------------------------------------------------- **Run at 135 V for 20 min. ==Results== [[Image:ZhangLab_2_2009-03-07_22hr_02min_crop.jpg]] [[Image:ZhangLab_2_2009-03-07_22hr_58min_crop.jpg]] *No bands showed up around 200bp for 3 different pairs of primer *Positive control showed clear band around ==Suggestion== *Taq 2X Mastermix is used instead of iQ supermix -Change to 2X iQ supermix *Annealing temperature 62C form the protocol is too high for the primers -Lower it to 50C (Do not change the condition) *Design primer for the target sequence without being methylated -Run PCR using both set of primers to confirm the effectiveness of methylation *Too much sample(80,000cells) resulting in incomplete bisulfite conversion -Use 10,000 cells next for conversion *PCR Preparation not done on the cooling block, and Zymo recommended using a hot-start ploymerase -Prepare reaction on cooling block and use a hot-start polymerase *Using 2% agarose gel to run gel electrophoresis should get better resolution *Use higher amount of ladder and load ladder next to the sample well.
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