AlanFung:LabNotes/EZ/2009-3-7

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EZ DNA Methylation Direct-GM20431 PCR analysis [edit]

Objective[edit]

  • Confirmation of the bisulfite conversion of GM20431 for 80,000 cells

Samples & Materials[edit]

  • 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[edit]

  • Cell Preparation
  • Bisulfite Conversion
  • PCR amplification
  • Agarose Gel Electrophoresis

Procedures[edit]

  • 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[edit]

  File:ZhangLab 2 2009-03-07 22hr 02min crop.jpg  File: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[edit]

  • 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.