I have run the TD-PCR in Bio-Rad Icycler IQ instrument but I can't get one band while I can get one band in conventional TD-PCR. May I have the advice from the experts here? Thank you.
Protocol:
Cycle 1: (1X)
Step 1: 95.0 °C for 10:00.
Cycle 2: (10X)
Step 1: 94.0 °C for 00:45.
Step 2: 55.0 °C for 00:30.
Increase set point temperature after cycle 2 by -1.0 °C
Step 3: 72.0 °C for 00:45.
Cycle 3: (35X)
Step 1: 94.0 °C for 00:45.
Step 2: 50.0 °C for 00:30.
Data collection and real-time analysis enabled.
Step 3: 72.0 °C for 00:45.
Cycle 4: (1X)
Step 1: 95.0 °C for 01:00.
Cycle 5: (1X)
Step 1: 55.0 °C for 01:00.
Cycle 6: (61X)
Step 1: 65.0 °C-95.0 °C for 00:10.
Increase set point temperature after cycle 2 by 0.5 °C
Melt curve data collection and analysis enabled.
Dear Tie
Plz use the condtions mentioned below
or consult the book attached below the pcr conditions
Stage 1:
95.0 °C for 1:00.
Stage 2: (40 Cycles)
Step 1: 95.0 °C for 00:30.
Step 2: 62.0 °C for 00:30.
Decrease set point temperature after cycle 2 by -1.0 °C (61, 60, 59, 58, 57, 56, 55, 54, 53, 52)
Step 3: 72.0 °C for 3:00.
Stage 3:
72.0 °C for 5:00.
Best Regards
Dear Afzal,
Thanks for the file and the suggestion. May I know where to detect SYBR green dye in real time? The decrease set point just for 10 cycles? What do you mean by the stage 2 of 40 cycles?
Sorry Dear
I sent to you notes from my PCR machine. I could not write it properly earlier. I am attaching my notes form my data book again.
I am sorry again becuase i did not see that you are doing touch down real time.
I usually do normal touchdown pcr, so it is my misunderstanding of the question.
I am sorry for causing the confusion.
Regards
Guys, I managed to optimised the real time TD-PCR protocol. Thanks for the advice and suggestions..
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