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Genotyping


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6 replies to this topic

#1 pmaj

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Posted 20 August 2009 - 02:05 PM

Hi all,

I am having trouble in doing my PCR for genotyping my mice. I have tried almost everything now. I bought a new Ex taq kit from Takara thinking my old polymerase was not working. But that did not help. I had a couple of Genomic DNA samples from my previous runs which had worked previously. I ran them with my current samples and I do not see anything. All I see is the ladder. The primer stocks have always been stored at -80 deg and the aliquoted working conc.s at -20 deg

I also called up the companyto double check, if the primers had gone bad since they were more than 18 months old, and the technical services rep said they should be fine.

I use .25microgm genomic DNA per sample in my PCR reaction. I measured the DNA concentrations from all the tail samples using the Nanodrop (which is supposed to be accurate!!). I tried measuring the concentrations again using a spectrophotometer in the lab (agilent 8453 spectrophotometer), and got slightly different measurements (10-25% difference). I used these new values and calculated the 250ng for the DNA samples to run my PCR, but I did not see anything, but the ladder.

Could someone tell me what is going wrong?

Thanks

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 20 August 2009 - 02:24 PM

Hi Pmaj,

The fact that your old DNA, which used to genotype fine, now does not work suggests to me that your problem is your primers. Primers that have been stored for 18 months can easily go bad, especially if you freeze and thaw them more than once, and if you store them at concentrations lower than 100 mM.

Here are some questions to consider for troubleshooting:

1. How did you extract your genomic DNA? Is it a crude prep or did you use a method that yields "pure" DNA and little to no protein (a kit for example)

2. How did you store your primers? Did you aliquot a permanent stock at 100 mM that you never thaw more than once to prepare your working stock (10 mM)?

In my experience the most common culprit for this kind of genotyping problem is the primers, assuming that you used fresh tissue and a standard DNA extraction procedure that yields half decent genomic DNA.

Edited by ivanbio, 20 August 2009 - 02:25 PM.


Ivan
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#3 Rsm

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Posted 20 August 2009 - 08:34 PM

Hi,
The obvious question: have you inactivated the Proteinase K? :lol:
Another try would be a positive control. For example, use the targeting vector mixed (or not mixed) with genomic DNA.
I hate genotyping...
Cheers,
Minna
I got soul, but I'm not a soldier

#4 pmaj

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Posted 21 August 2009 - 08:05 AM

Hi Pmaj,

The fact that your old DNA, which used to genotype fine, now does not work suggests to me that your problem is your primers. Primers that have been stored for 18 months can easily go bad, especially if you freeze and thaw them more than once, and if you store them at concentrations lower than 100 mM.

Here are some questions to consider for troubleshooting:

1. How did you extract your genomic DNA? Is it a crude prep or did you use a method that yields "pure" DNA and little to no protein (a kit for example)

2. How did you store your primers? Did you aliquot a permanent stock at 100 mM that you never thaw more than once to prepare your working stock (10 mM)?

In my experience the most common culprit for this kind of genotyping problem is the primers, assuming that you used fresh tissue and a standard DNA extraction procedure that yields half decent genomic DNA.


Hi Ivan,

I did the crude extraction method. the tail snipping and then digestion with Proteinase K. The reason I do not use a kit or some other form of extraction,since I have a really large colony of mice and I need to keep up with their genotypes. I had been using the crude way of extraction and it used to work until now :-(

I think it must be the primers also. I made a working conc. of 20micro M and stored them in small aliquots. I have freeze thawed them a couple of times:-(. The stock is still in the -80 freezer. I think I will thaw it and make fresh working conc. primers. or I think I will order fresh primers again!

Thanks for your 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

#5 pmaj

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Posted 25 August 2009 - 10:48 AM

Hi,
The obvious question: have you inactivated the Proteinase K? :lol:
Another try would be a positive control. For example, use the targeting vector mixed (or not mixed) with genomic DNA.
I hate genotyping...
Cheers,
Minna


Tell me about it. I hate genotyping too:-(

How could I have inactivated the Proteinase K? the DNA samples have always been at 4Deg C and When I use them I always place them on ice.

thanks

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 ivanbio

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Posted 28 August 2009 - 06:35 AM

I agree with Minna that your proteinase K could be still active, which would chew up your Taq and lead to no PCR. Typically after you perform the proteinase K digestion step, you can incubate your samples at ~95oC for a few minutes (I do it for 3 minutes). Some people think this is not essential if your next step is an ethanol precipitation step (which gets rid of the proteinase K), but I think it is better to be safe than sorry. My gut feeling, if this is indeed your problem, is that for whatever reason your proteinase K is pretty resilient and in this particular batch some of it is surviving through the extraction step. Plus, even at 4oC a little bit of proteinase K will slowly chew away your DNA.

I do agree that ordering new primers is a good idea. In my experience primers that have been thawed as little as 2 or 3 times, can already start to degrade, especially when they are stored at low concentrations (~10 uM). What I do is store aliquots of a 100 uM stock and only prepare 10 uM aliquots, which I too store frozen, when I need them (and only keep enough for up to 6 months of usage).

Minna and pmaj: If you hate genotyping, have you ever considered outsourcing? There are companies that will run your genotyping for you at a pretty reasonable price ($5.50 per sample) and you do not have to worry about the assay not working, or spending a good portion of your day doing it.

Ivan
Carlsbad, CA

#7 pmaj

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Posted 29 August 2009 - 10:27 AM

I agree with Minna that your proteinase K could be still active, which would chew up your Taq and lead to no PCR. Typically after you perform the proteinase K digestion step, you can incubate your samples at ~95oC for a few minutes (I do it for 3 minutes). Some people think this is not essential if your next step is an ethanol precipitation step (which gets rid of the proteinase K), but I think it is better to be safe than sorry. My gut feeling, if this is indeed your problem, is that for whatever reason your proteinase K is pretty resilient and in this particular batch some of it is surviving through the extraction step. Plus, even at 4oC a little bit of proteinase K will slowly chew away your DNA.

I do agree that ordering new primers is a good idea. In my experience primers that have been thawed as little as 2 or 3 times, can already start to degrade, especially when they are stored at low concentrations (~10 uM). What I do is store aliquots of a 100 uM stock and only prepare 10 uM aliquots, which I too store frozen, when I need them (and only keep enough for up to 6 months of usage).

Minna and pmaj: If you hate genotyping, have you ever considered outsourcing? There are companies that will run your genotyping for you at a pretty reasonable price ($5.50 per sample) and you do not have to worry about the assay not working, or spending a good portion of your day doing it.

:lol:

Hi Ivan and Minna,

So I ordered new primers and reconstituted them in 10mM Tris. My working conc. for the primers is 20uM. I also prepared working concs. of primers from my frozen stock. I used both these sets of primers (old and new), with my DNA samples which had worked for me before. so I ran these samples in duplicate, one using the old primers (freshly made from frozen stock) and Newly ordered primers. I did NOT see anything. I just saw the ladder.

I used to use Etbr in my agarose gel, which I am not very comfortable with, so I ordered a non-mutagenic dye, EZ vision from Midsci. I only saw the ladder and no other bands. I am soaking my gel in Etbr soln. prepared in 1x TBE, to see if I have any bands.

Also all my genomic DNA samples were quantified Using the nanodrop instument. I took some samples and ran them manually through the spectrophotometer in the lab and I got slightly different results. Then I ran 2 different sets of PCR reactions using the two different conc, for the same samples, but I did not see anything. Previously, I would just measure the DNA conc using the spectrophotometer by running the samples in triplicate and taking an avg. and I would see fantastic bands on my gels.

I have gone ahead and ordered the Denville Tissue DNA extraction kit. But I have almost 80 samples which need to be genotyped. I cannot outsource them cause it will cost us a pretty penny:-(

I am at a complete loss what to do next!

Sincerely,

Pmaj :lol:

Edited by pmaj, 29 August 2009 - 10:28 AM.

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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