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TOUCH DOWN PCR NEEDS EXPLANATION


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3 replies to this topic

#1 Microbes in action

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Posted 20 February 2011 - 05:08 AM

I have a good PCR products with a good sensitivity with this T.D.PCR cycle.....

THIS WORKS GOOD
95c - 6mins

95c-50s
67c-1min - 3 cycles
68c-2mins

95c-50s
67c-1min - 8cycles (reducing cycle, 1c decreament in each cycle, so it reaches 60c)
68c-2mins

95c-50s
67c-1min - 29 cycles (normal)
68c-2mins

72c-5mins

BUT THIS DOES NOT WORK.....
95c - 6mins

95c-50s
67c-1min - 3 cycles
68c-2mins

95c-50s
67c-1min - 8cycles (reducing cycle, 1c decreament in each cycle, so it reaches 60c)
68c-2mins

95c-50s
60c-1min - 29 cycles (normal)
68c-2mins

72c-5mins

CAN SOME ONE EXPLAIN WHAT IS HAPPENING IN THESE TWO REACTIONS.....& I USED SAMPLES OF DNA DILUTION, THE SENSITIVITY BETWEEN THESE TWO REACTION IS ATLEAST ONE LOG.THE FIRST REACTION GAVE ME A SENSITIVITY OF 10^6 & THE SECOND ONE CAN COME UP TO ONLY 10^4

PLEASE THROW SOME DISCUSSION ON THIS.

FIND PICS.

#2 seanspotatobusiness

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Posted 23 February 2011 - 05:23 AM

I have a good PCR products with a good sensitivity with this T.D.PCR cycle.....

THIS WORKS GOOD
95c - 6mins

95c-50s
67c-1min - 3 cycles
68c-2mins

95c-50s
67c-1min - 8cycles (reducing cycle, 1c decreament in each cycle, so it reaches 60c)
68c-2mins

95c-50s
67c-1min - 29 cycles (normal)
68c-2mins

72c-5mins

BUT THIS DOES NOT WORK.....
95c - 6mins

95c-50s
67c-1min - 3 cycles
68c-2mins

95c-50s
67c-1min - 8cycles (reducing cycle, 1c decreament in each cycle, so it reaches 60c)
68c-2mins

95c-50s
60c-1min - 29 cycles (normal)
68c-2mins

72c-5mins

CAN SOME ONE EXPLAIN WHAT IS HAPPENING IN THESE TWO REACTIONS.....& I USED SAMPLES OF DNA DILUTION, THE SENSITIVITY BETWEEN THESE TWO REACTION IS ATLEAST ONE LOG.THE FIRST REACTION GAVE ME A SENSITIVITY OF 10^6 & THE SECOND ONE CAN COME UP TO ONLY 10^4

PLEASE THROW SOME DISCUSSION ON THIS.

FIND PICS.



Without knowing the sequences of the primers or whether there is mispriming evident on your gels, I'm guessing that a) there's mispriming at the lower temperature or B) there are primer dimers forming at the lower temperature. Either way, it looks like you might as well forget the touchdown part and just do 35 cycles at the higher temperature.

#3 Microbes in action

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Posted 23 February 2011 - 09:30 AM

Thanks much for your reply.

please find the attached pictures.

samples are DNA dilutions.

Attached Thumbnails

  • REGULAR METHOD.JPG
  • TOUCH DOWN METHOD.JPG


#4 chimaera

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Posted 24 February 2011 - 06:32 AM

Depending on the enzyme, the polymerase can work at 67^C as well, which means a longer extension in the first protocol.




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