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smearing below band of interest - (Jun/30/2012 )

i am doing touchdown pcr with the following condition:

98'C 30 sec initial denaturation

10 cycles:
98'C 5 sec denaturation
60'C 5 sec annealing
72'C 5 sec extension

25 cycles:
98'C 5 sec denaturation
59'C 5 sec annealing
72'C 5 sec extension

72'C 2 min final extension

my band of interest is 200bp
i got the band
but there are smearing below the band:
Attached File

what has gone wrong and how should i rectify the problem?

FYI, my pcr master mix contain 18.5ul master mix, 0.5 ul forward primer, 0.5 ul reverse primer, 0.5 ul DNA.

-jamestoon1-

Given the 98 denature, I assume you are using Phusion. Phusion master mixes are usually 2x master mixes. You should double check to make sure you are not missing the added water.

-phage434-

How much of your PCR volume did you load on the gel? It looks very overloaded to me. The smearing below your product could be primer dimer.

Just out of interest, can I ask what the rationale for using those cycle conditions is? It seems odd to me to have two annealing temperatures that are SO close.

-leelee-

phage434 on Sun Jul 1 12:07:28 2012 said:


Given the 98 denature, I assume you are using Phusion. Phusion master mixes are usually 2x master mixes. You should double check to make sure you are not missing the added water.


Yes, it's Phusion. It was a typographical error. What I used was 10ul master mix plus 8.5ul water.

-jamestoon1-

leelee on Sun Jul 1 14:23:03 2012 said:


How much of your PCR volume did you load on the gel? It looks very overloaded to me. The smearing below your product could be primer dimer.

Just out of interest, can I ask what the rationale for using those cycle conditions is? It seems odd to me to have two annealing temperatures that are SO close.

I loaded 3ul.
60'C is the highest achievable temp. I mean, after 60'C, i can no longer get any pcr product. so i chose to use tat one as the starting temperature.
and i chose 59'C because from my optimization experiments, 59'C can result in multiple bands, so i would not go below that temp.
there should be no problem with primer design. i have BLASTed and have checked with a few softwares. Moreover this is the primer pair obtained from literature. A lot of people have used this primer pair.

-jamestoon1-

If you get a product at 60C, why are you bothering with different temperatures?

Given that you are loading 3ul of a 20ul reaction and getting that much DNA in your lanes, I think that you are using too much template and that is what you are seeing on your gel. There is a huge amount of DNA up near the well too.

My advice is to do a dilution series of your template (say a 1/10, 1/100, 1/1000 and 1/10000) then run the PCR again.

-leelee-