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Running Primers on Agarose Gel

primers agarose gel

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6 replies to this topic

#1 senataxin

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Posted 21 March 2012 - 05:43 AM

Hi Everyone,

I run my stock primers on 1.5% agarose gel and saw that the difference in intensities of forward and reverse primers is huge.
The stock concentrations of both forward and reverse are 200pmol/ul.

So what might be the reason of this difference? Something with calculation?

Thank you,
Senataxin

#2 Trof

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Posted 21 March 2012 - 08:31 AM

What length are your primers? Is one markedly longer than another? Because same molarity doesn't mean same mass, intensity on gel is proportional of overal bp present.
Why are you running them on gel anyway, your PCR is not working?

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

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#3 senataxin

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Posted 21 March 2012 - 11:15 AM

Both forward and reverse are 21 bp length.

My PCR is not working; I can not see my band, there is only a few weak non-specific bands. My PI wants to check whether my primers are degraded. Luckily they are not, but somehow they have different intensities on gel.

Thank you for your answer Trof.

#4 Trof

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Posted 22 March 2012 - 03:43 AM

I wouldn't be sure it's possible to see degradation of 20bp primers on agarose gel. You would need acrylamide.
Do you have a picture of the gel?

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 senataxin

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Posted 22 March 2012 - 03:54 AM

I attached the gel image, thank you!!!

Attached Thumbnails

  • gel.png


#6 Trof

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Posted 22 March 2012 - 06:02 AM

Don't see any size marker. But the primer on the right looks more like degraded, that can be the cause of lower intensity. But that's hard to say, you see, It's a pretty wide band on the left, that could be caused by huge quantity, but also different-sized fragments. Bands this short on agarose are always a bit smeary though.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#7 senataxin

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Posted 22 March 2012 - 06:27 AM

We are going to try a few optimization, and going to change the primer stocks we are using if PCRs are not still working. Hope that works...





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