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PCR - No Band formation


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#1 aks

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Posted 04 August 2010 - 02:34 PM

Hi,
I used the PV92 Informatics Kit from BIO-RAD to amplify the samples in the PCR. I followed the protocol as mentioned in the manual. After i ran the samples in the agarose gel, I could see only bands from the Molecular Mass Ladder(MMR), and migration of loading dye from the other wells. Is there any way I can check till what stage the protocol works and any idea as to why it is not working?
I have attached a picture of the gel after it was stained. The right most well was loaded with MMR (the one that has bands)
Thanks in advance for your help !!!

IMG_0411.JPG

Edited by aks, 04 August 2010 - 02:54 PM.


#2 bob1

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Posted 04 August 2010 - 05:53 PM

Seeing as you are a learner at PCR... What do you think could have gone wrong? Most people on here won't help you much unless you help yourself first.

#3 jaya2020

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Posted 08 August 2010 - 11:25 AM

Hi,
I used the PV92 Informatics Kit from BIO-RAD to amplify the samples in the PCR. I followed the protocol as mentioned in the manual. After i ran the samples in the agarose gel, I could see only bands from the Molecular Mass Ladder(MMR), and migration of loading dye from the other wells. Is there any way I can check till what stage the protocol works and any idea as to why it is not working?
I have attached a picture of the gel after it was stained. The right most well was loaded with MMR (the one that has bands)
Thanks in advance for your help !!!

IMG_0411.JPG


hi

This is very simple as your gel looks. The annealing has not gone well. Check your annealing temperature. In PCR it is the annealing temperature which is important. How do you set the annelaing temperature. One simple ways is just add the annelaling temp that is given in the data sheet that comes with the primer, divide by 2 and substract 2 from it. Generally this will work. If not increase or decrease the temperature by one degree. If you have a gradient PCR machine, do it for a range of temperature. Run on a gel. This will give you the correct annealing temp. Good luck

#4 aks

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Posted 09 August 2010 - 07:36 AM

Thank you so much Jaya2020! I checked the block temperature and I realized that it was not calibrated properly. I programed the cycles for the temperature that was mentioned in the manual. Will be running it sometime soon. Hope it works! :)




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