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