AlanFung:LabNotes/EZ/2009-3-10

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EZ DNA Methylation Direct-GM20431 PCR analysis [edit]

Objective[edit]

  • Confirmation of the bisulfite conversion of GM20431 for 1,000 cells
  • Continuation of lowering cell number
  • Increase low mass ladder volume to 2uL for a better reference


  • 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     0     0 
  gDNA                0.5    0.5    0.5   0.5   0
  RNAse free H20      7.5    7.5    7.5   15.5  16     
                      -----------------------------
                                                40uL
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
  • 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     2      0
0.5X TBE Buffer  0     4     4     4     4    4     3      0
6X Loading Dye   0     4     4     4     4    4     3      0
-------------------------------------------------------------
    • Run at 135 V for 20 min.

Results[edit]

 File:ZhangLab 2 2009-03-10 11hr 49min crop.jpg
 
*Clear bands showed up around 200bp for primer CHR22 only
*Positive control showed dimmer band around than previous PCR
*Negative control still showing up and its brighter than usual, it is even brighter than the +ve control
*Ladder is showing up better but still not clear enough

Troubleshoot[edit]

*Run a side by side bisulfite conversion of  concentration of gDNA and 
*Samples <2000 cells should be treated with less M-Digestion Buffer according to the Zymo protocol
*Increase the ladder volume to 3uL
*Increase the concentration of the agarose gel to 1.5%-2%