PCR faint bands under UV light - (Feb/20/2008 )

Hello,

I am having a problem with PCR amplification.

I obtained DNA from blood samples and I am amplifying a certain exon region of a gene. My problem is that after I do the PCR, I get very faint bands under UV light and I cannot do gel extraction.

I use 7 ul DNA, 3 ul of primer, 50ul of Hot star master mix and 40ul Water. The Tm of my primers are 53.4 and 56.9 degrees. I have tried amplifications at 52, 54 and 55 degrees. As I said, under UV light, I do get some bands, but they are too faint to do gel extraction.

I was hoping to get some ideas or suggestions about what to do?

Thanks.

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100 ul is quite a large volume for a PCR reaction. How much of the volume are you loading on the gel?

Also, what is the cycle number?

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Try to put your PCR product into a TOPO vector. Then you can either directly cut it out of the TOPO vector or repeat the PCR using the TOPO clone as template to get more product.

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I am trying to load the entire 100ul on the gel but some of it leaks out.

48 cycles is what I am using.

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Try lowering your annealing tempurature.... it should be about 5 degrees less than the primer Tm.

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Sorry to ask more questions... but what is the size of your PCR product, and what will you use the DNA for after gel extraction?

Some observations... 48 cycles is too many, I wouldn't do more than 40. If you need to have the correct sequence in the final product, you should use Hi Fi taq and limit your cycle number to about 25. If it is a very large region, you could try to amplify it in two pieces.

If you have trouble with leaking out of gel, make a larger well by taping two wells together. You could do several PCR reactions and combine them for the gel extraction, but check the maximum weight of agarose you can have in the gel extraction kit.

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Thanks for your replies-

the product is 480bp. I have found a mutation in a large population of samples and I have to sequence it to verify it. So I want to gel extract my DNA and do direct sequencing.

I will try the PCR with 40 cycles and will also try several PCR reactions and then pool them together.

Thanks.

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What concentration of primers and dNTPs are you using? How large if the exon? and how long is your extension time?

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Hi,

I did lower the annealing temperature to 52 degrees and I did 3 PCR reactions at 52, 54 and 55 degrees. All the 3 looked the same ( faint bands) under UV light. So I think the annealing temperature will be fine at any of these temperatures.

Thanks.

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Three things you can do:

1. Add an additional 4 mM MgCl2 to your reaction. Magnesium strengthens the annealing of primers to the template and will increase the brightness of your products.

2. Check the GC% of your amplicon. If it is 55% or greater do a betaine gradient (0.5, 1.0, 1.5, 2.0 M). Betaine usually helps to amplify high GC% targets. It equalises the strength of GC and AT bonds so that the target dissociates more easily.

3. Design some new primers so that they anneal to close but different regions of your target. Importantly make sure the new primers have a different 3` end. Sometimes a particular primer for some reason or another does not work well and prevents the whole PCR from happening. A different primer can make all the difference. You never know which primer is not working, so that's why i design a new primer at each end. You can try all the combinations of primers (old and new) to see which ones work and which ones fail, and/or pool them all into the one reaction. Personally, i like primers with a Tm of 70C to increase the specificity of annealing, but if you are getting no secondary bands in your PCR you don't need to worry about that.

Good luck,
Rob

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