5 replies to this topic

[micki]

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!

[nightingale]

Hello Micki

make sure of your primers ...

[GNANA]

Do a primer blast (NCBI) and check the possibility of amplifying your target and the suspect....

[micki]

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?

any idea about this?

[micki]

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

[phage434]

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