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questions about semi-quantitive PCR


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#1 tantao

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Posted 10 August 2009 - 05:15 AM

Dear everyone, I have got a weird results from semi-quantitive PCR.
I have two samples(A and B), and I amplified the GAPDH in order to find a approprate amount of templete to amplify other genes. But I got a weird result. I used 1ul templete B to run the PCR and 1.3ul, 1.4ul and 1.5ul templete A(60℃, 24cycle). I made the buffer mixture and aliquot it. But amplification efficiency is not the same in the templete B . Moreover in templete A, 1.3ul templete was amplified more than 1.4ul. I have repeated this for 2 times, and got same result every time. I do not know why,and now I do not know choose how many templete A to amplify other genes. Please help me.:(

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#2 uvbox

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Posted 15 August 2009 - 11:49 PM

Dear everyone, I have got a weird results from semi-quantitive PCR.
I have two samples(A and B), and I amplified the GAPDH in order to find a approprate amount of templete to amplify other genes. But I got a weird result. I used 1ul templete B to run the PCR and 1.3ul, 1.4ul and 1.5ul templete A(60℃, 24cycle). I made the buffer mixture and aliquot it. But amplification efficiency is not the same in the templete B . Moreover in templete A, 1.3ul templete was amplified more than 1.4ul. I have repeated this for 2 times, and got same result every time. I do not know why,and now I do not know choose how many templete A to amplify other genes. Please help me.B)


Hey, dont know if you're new to PCR and gel loading etc but i am and this is kind of what happened to me as well.
I know this might sound stupid, but the way you are pipetting makes a big difference and might explain why ur sample B (all 1ul) are not producing the same band intensities.

As you're working with 1ul, this is actually tiny volume and for beginners (like me) its easy to lose some template due to bad pipetting. Losing just a tiny bit in the pipette tips (0.1-0.2ul) would mean losing 10-20% of ur sample, so yea~

So..i guess ur 1.3ul being more than 1.4ul could probably also be explain by what i've said above..(theres only 0.1ul difference! so its easy to lose some template)

Hope this helps!

Edited by uvbox, 15 August 2009 - 11:52 PM.


#3 Penguin

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Posted 16 August 2009 - 06:45 AM

Dear everyone, I have got a weird results from semi-quantitive PCR.
I have two samples(A and B), and I amplified the GAPDH in order to find a approprate amount of templete to amplify other genes. But I got a weird result. I used 1ul templete B to run the PCR and 1.3ul, 1.4ul and 1.5ul templete A(60℃, 24cycle). I made the buffer mixture and aliquot it. But amplification efficiency is not the same in the templete B . Moreover in templete A, 1.3ul templete was amplified more than 1.4ul. I have repeated this for 2 times, and got same result every time. I do not know why,and now I do not know choose how many templete A to amplify other genes. Please help me.B)


Is this a good way to do semi-quantitative PCR? As uvbox said pipetting errors are huge with such small volumes, but also after 24 cycles of PCR are you really going to see differences in copy number when you are now working with millions of amplified copies??

The way I do semi-quantitative PCR is to set up a 50ul reaction and then take 5 ul samples every 5 cycles for upto 30 cycles - I then use densitometry to draw a graph (similar to the way qPCR draws curves)

P




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