AlanFung:LabNotes/EZ/2009-3-21

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PCR of EZ DNA Methylation Direct-GM20431 100 & 10 cells with equal amount Jurkat gDNA[edit]

Objective[edit]

Gel electrophoresis to analyize the methylation of DNA

Procedures[edit]

Gel 1 100 cells
Well             1     2     3     4     5    6     7      8
-----------------------------------------------------------------
Content          Blank AA    AB    AC    CA   CB    CC     Ladder
Sample           0     6     6     6     6    6     6      3
0.5X TBE Buffer  0     4     4     4     4    4     4      3
6X Loading Dye   0     4     4     4     4    4     4      3
-----------------------------------------------------------------
Total                                               14uL   9uL
Volume to be aspirated                              13.4uL 8.4uL
Gel 2 10 cells
Well             1     2     3     4     5    6     7      8
-----------------------------------------------------------------
Content          Blank BA    BB    BC    DA   DB    DC     Ladder
Sample           0     6     6     6     6    6     6      3
0.5X TBE Buffer  0     4     4     4     4    4     4      3
6X Loading Dye   0     4     4     4     4    4     4      3
-----------------------------------------------------------------
Total                                               14uL   9uL
Volume to be aspirated                              13.4uL 8.4uL


  • Run both gels at the same time at 135 V for 20 min.

Results[edit]

File:ZhangLab 2 2009-03-21 23hr 11min crop.jpg

No bands showed up at 200bp for both 100 and 10 cells, while the equilvalent amount of Jurkat gDNA did show positive results.

Suggestion[edit]

  • Repeat gel electrophoresis with 2nd set of samples
  • Increase sample to 10uL
  • No need to dilute loading dye with TBE buffer, dilue with samples


  • Repeat experiment with 1000 cells as +ve control
  • Mix the binding buffer with the samples by inverting the tube couple of times instead of repeat pipetting.
  • Reduce washing step before elution to one time could possibly generate a higher yield


  • Perform MDA WGA right after CT conversion to increase both quantity and quality of the bisulfite converted DNA