primer design

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winas
winas's picture
primer design

Hi

i am struggling to design my primers for TAF15-201

i am supposed to do it for 2 consecutives exons, but i don't know what i am suppose to do with introns
do i need the full intronic sequence?
how do i link both exonic sequences?

Then once in my primer design tool, and knowing that i need to amplify at least 30bp upstream and downstream of the exons encoding TAF15-201
What are the ranges for my forward and reverse primer what is my pcr size ?

where do my primers sit in my sequence ?

any help would be appreciated

Ivan Delgado
Ivan Delgado's picture
Hi winas,

Hi winas,

If I understand correctly you are trying to PCR amplify each one of your two exons, likely to clone them for expression or something else. Amplifying 30 bp at both ends of the exons is one way to amplify the whole exons since that way you have some extra DNA at both ends for cloning purposes. 

As for why you are having problems designing the primers, I am not sure what your problems are. All you need to do is obtain the genomic sequence of your gene, identify the ends of each exon, and the design primers flanking your exons. Your primers simply sit outside your exons, in the intron sequence, around 30 bp of the exon's ends. As for the ranges of the primers, typically a good primer can be between 20 to 28 bp. 

If I am missing something please let me know. 

winas
winas's picture
Thanks for your reply

Thanks for your reply

Yes i am trying to amplify two exons to work on gene expression

my problem being this is the first time i am using blast and i am not sure how to set it up correctly
i don't know what pcr size i must use and what are the ranges for my forward and reverse primers

Everything i ve tried was unsuccessful

Ivan Delgado
Ivan Delgado's picture
Hi winas,

Hi winas,

Unfortunately explaining how to use BLAST here would be somewhat of a challenge. I would recommend asking someone at your institution. I've used BLAST for a very long time and I really cannot think of anything special other than simply selecting from the drop down menus the type of DNA (genomic) and the organism you are interested in.

As for the size of the PCR products you want to amplify, that is dependent on the size of the exons. If the exons are 500 bp then your PCR product will be around that size. 

Sorry but I cannot think of anything else I can do to help you out.

winas
winas's picture
Thanks for your time Ivan

Thanks for your time Ivan
i am starting from there