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Genotyping


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6 replies to this topic

#1 pmaj

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Posted 03 August 2009 - 08:21 AM

Hi,

I am trying to genotype mice by using Tail DNA, and amplifying the gene of interest. I am using 250ng DNA per sample. I use 1% agarose gel with Ethidium bromide. I have done this a lot of times, but Off late I havn't been able to see anyting. I amplified the samples again and ran a ladder and I do not see the ladder as well. What could be going wrong?

Confused! :(

Pmaj
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
~Judah Folkman

#2 ivanbio

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Posted 03 August 2009 - 08:39 AM

Hi pmaj,

The fact that you do not even see your ladder suggests to me that your problem is not your PCR assay but gel imaging. I doubt your ethidium bromide went bad, but that is a possibility. Another issue could be your gel documentation system or camera. Typically these instruments comes with a fluorescent ruler; use it to make sure your filter/detection device is working the way it should.

On the PCR side, it is not uncommon for a genotyping PCR assays to stop working. One reagent may have gone bad or your latest DNA extraction did not work as it should have. One good thing to do to make sure this is not the case is to get all fresh/new reagents and start over.

Good luck

Ivan
Carlsbad, CA

#3 cellcounter

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Posted 03 August 2009 - 08:44 AM

Hi pmaj,

The fact that you do not even see your ladder suggests to me that your problem is not your PCR assay but gel imaging. I doubt your ethidium bromide went bad, but that is a possibility. Another issue could be your gel documentation system or camera. Typically these instruments comes with a fluorescent ruler; use it to make sure your filter/detection device is working the way it should.

On the PCR side, it is not uncommon for a genotyping PCR assays to stop working. One reagent may have gone bad or your latest DNA extraction did not work as it should have. One good thing to do to make sure this is not the case is to get all fresh/new reagents and start over.

Good luck


As said earlier, you must see the ladder first. There is no point in worrying about PCR when EtBr-agarose gel imaging does not work.

#4 perneseblue

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Posted 03 August 2009 - 08:29 PM

Since the DNA ladder can't be seen, do check your gel imagining apparatus. The UV lamp needs to be replaced every so often. Make sure the camera is still okay. Best to get hold of a second gel imaging system and run a test gel containing only a DNA ladder.

But also check your agarose gel. Are you using new gel? If the gel is heavily contaminated/old/recycled, the DNA can degrade while on the gel.

P.S also make sure the electrodes on your gel electrophoresis box is on the right way and not reversed.
May your PCR products be long, your protocols short and your boss on holiday

#5 pmaj

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Posted 05 August 2009 - 08:09 AM

Hi pmaj,

The fact that you do not even see your ladder suggests to me that your problem is not your PCR assay but gel imaging. I doubt your ethidium bromide went bad, but that is a possibility. Another issue could be your gel documentation system or camera. Typically these instruments comes with a fluorescent ruler; use it to make sure your filter/detection device is working the way it should.

On the PCR side, it is not uncommon for a genotyping PCR assays to stop working. One reagent may have gone bad or your latest DNA extraction did not work as it should have. One good thing to do to make sure this is not the case is to get all fresh/new reagents and start over.

Good luck


Hi Ivan,

I did order a new polymerase kit, new agaroase. Hopefully my DNA samples are not bad!

Thanks for the input

Pmaj
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
~Judah Folkman

#6 pmaj

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Posted 05 August 2009 - 08:11 AM

Since the DNA ladder can't be seen, do check your gel imagining apparatus. The UV lamp needs to be replaced every so often. Make sure the camera is still okay. Best to get hold of a second gel imaging system and run a test gel containing only a DNA ladder.

But also check your agarose gel. Are you using new gel? If the gel is heavily contaminated/old/recycled, the DNA can degrade while on the gel.

P.S also make sure the electrodes on your gel electrophoresis box is on the right way and not reversed.


Hi Perneseblue,

I did order new agarose. I think I was using old gel:-(

about the electrodes, that is a good tip. I did that once long ago and am always careful about that ever since:-)

Thanks for the input

Pmaj
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
~Judah Folkman

#7 pmaj

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Posted 06 August 2009 - 03:04 PM

Hi pmaj,

The fact that you do not even see your ladder suggests to me that your problem is not your PCR assay but gel imaging. I doubt your ethidium bromide went bad, but that is a possibility. Another issue could be your gel documentation system or camera. Typically these instruments comes with a fluorescent ruler; use it to make sure your filter/detection device is working the way it should.

On the PCR side, it is not uncommon for a genotyping PCR assays to stop working. One reagent may have gone bad or your latest DNA extraction did not work as it should have. One good thing to do to make sure this is not the case is to get all fresh/new reagents and start over.

Good luck


As said earlier, you must see the ladder first. There is no point in worrying about PCR when EtBr-agarose gel imaging does not work.


I tried using someone else's Taq, and used fresh 1%agarose solution . i see the ladder, but am not seeing my bands. what could be going wrong now?

:-(
There is a fine line between persistence and obstinacy. I have come to realize the key is to choose a problem that is worth persistent effort.
~Judah Folkman




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