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sequencing result doesn't match my gene....

sequencing snp

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5 replies to this topic

#1 micki

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Posted 09 May 2012 - 08:21 PM

Hi all,

the project that i am currently doing is about the genotyping of SNP (human) by using PCR-RFLP. i am facing some problems with the sequencing result (using PCR products of 200 bp):
1) My sequencing result doesn't show high % identity with the gene that i am studying but show high identity with other gene after i Blastn the result.
2) in the result, the sequence should contain the primers and SNP right? but i couldn't locate any of them in the result that i obtained.. how could this happened?
3) the sequencing chromatogram doesn't show any multiple/ overlapping peaks.. the peaks are regular and nicely separated.

As the purpose of my study is to genotype the SNP and confirm it by performing sequencing, the sequencing result that i obtained doesn't appear to be as what i expected...kinda frustrated..

any idea? anyone can plz help me?

tq very much!

#2 nightingale

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Posted 10 May 2012 - 12:09 AM

Hello Micki :)

make sure of your primers ...
" The more you learn, the more you realize how little you know ... "

#3 GNANA

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Posted 10 May 2012 - 01:33 AM

Do a primer blast (NCBI) and check the possibility of amplifying your target and the suspect....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#4 micki

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Posted 10 May 2012 - 04:23 AM

my primers supposed to amplify the sequence that i want. but in my sequencing result, the primer sequence even didn't appear in it.. is it possible to get such result?
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any idea about this?

#5 micki

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Posted 10 May 2012 - 04:26 AM

the length of my sequencing result is even longer than the pcr product length... very confuse...Posted Image

#6 phage434

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Posted 10 May 2012 - 04:29 AM

You probably have someone else's sequencing results. Or your samples were mixed up prior to sequencing.





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