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PCR Help

PCR Help - Molecular Biology Techniques

PCR Help - Molecular Biology Forum. Includes forums for common molecular biology techniques.


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  #1  
Old 07-12-2013, 09:36 PM
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Unhappy PCR Help



I need some ideas on what has happened to my PCR. I have been doing genotyping on a mouse and all I am getting are smears. I have isolated new DNA, ordered new Taq, buffer, and MgCl2. I can't think of anything else to change and I really need to get this genotyping done ASAP. This genotyping has previously worked, which makes me even more confused.
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Old 07-15-2013, 02:34 PM
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Default Re: PCR Help

Are the primers you used freshly prepared? I mean were they prepared from the main stock (where the lyophilized primers where diluted) for this new genotyping?

Why am I telling you this? Because primers tend to gradually degrade after repeated freeze-thaw cycles.

I suggest you prepare new primers from stock and repeat the PCR. I think primers might be your issue since you mention you get smears, which I believe are template DNA.

Check it and post your results to congratulate you or for further guide in case it does not work.
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Old 07-16-2013, 03:57 PM
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Default Re: PCR Help

I've diluted new lysophilized primers and I'm still getting streaking. I've attached a picture of the gel. I'm beginning to think the DNA extraction kit I have may be contaminated or something. I can't think of anything else that could cause this.
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File Type: jpg 7-16-13 genotype.jpg (5.1 KB, 2 views)
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Old 07-16-2013, 06:37 PM
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Default Re: PCR Help

Do you quantify DNA before PCR? That smear looks just like DNA to me, which means you might have added a bit too much template to your PCR reaction thus inhibiting the polymerase.

You should try diluting your samples before PCR. For the looks of the smear (very solid) I suggest you start at 1:10, I mean mix 1 uL of sample DNA with 9 uL of water prior to PCR reaction. From this 1:10 DNA mix you can take whatever volume you need for you PCR.

Good luck.
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  #5  
Old 07-17-2013, 01:25 PM
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Default Re: PCR Help

The DNA is diluted to 1ng/ul with a 4ul load in a 20 ul reaction. I rechecked it yesterday and my 260/280 is perfect so I don't think it is protein contamination. My supervisor suggested that it may be ethanol contamination from the DNA extraction process and suggested evaporating and re-suspending in water. Trying that today.
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Old 07-17-2013, 09:14 PM
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Default Re: PCR Help

Update: It finally worked. I still got a little streaking but I got very clear bands in the right place. Thank you so much for your help.
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