Hi Raha

if you mean the amplicon size that your primers will generate then the best way is to use NCBI BLAST: this will identify the region of sequence your primers bind to and in doing so the amplicon size

1. You simply submit your sequence and it will map it to the appropriate coding sequence in the Genbank database 

2. You then want to do the same for you other primer and make sure your principal hot is the same coding sequence 

3.  Finally export that sequence into something like word and then use the search function to map the sequence of primer letters back onto to your Genbank coding sequence.  Remember:  you reverse primer is actually a reverse complement of the actual target sequence so you need to convert the primer sequence into the actual target sequence using an 'reverse compliment ' tool you can easily find in the internet

alternatively you can map primer sequences to the appropriate coding sequence and deduce amplicon size more directly using primer BLAST 

Find attached a link to primer blast and also Oligo calculator: the latter will enable you to BLAST primers to map to parent sequence and also reverse compliment your reverse primer so you can match this to actual amplified sequence

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.basic.northwestern.edu/biotools/oligocalc.html

For finding gene size from primer, I am sending mouse albumin rRNA sequence. Here size of the PCR product is 297 bp. 

Same you can try to do with your Alkaline Phosphatase gene.

regions between two primers is expected size of PCR product. 

Hope this will help. .

thank but how did you get that sequence. could you send me the steps please

Thanks again

Hi Raha

if you mean the amplicon size that your primers will generate then the best way is to use NCBI BLAST: this will identify the region of sequence your primers bind to and in doing so the amplicon size

1. You simply submit your sequence and it will map it to the appropriate coding sequence in the Genbank database 

2. You then want to do the same for you other primer and make sure your principal hot is the same coding sequence 

3.  Finally export that sequence into something like word and then use the search function to map the sequence of primer letters back onto to your Genbank coding sequence.  Remember:  you reverse primer is actually a reverse complement of the actual target sequence so you need to convert the primer sequence into the actual target sequence using an 'reverse compliment ' tool you can easily find in the internet

alternatively you can map primer sequences to the appropriate coding sequence and deduce amplicon size more directly using primer BLAST 

Find attached a link to primer blast and also Oligo calculator: the latter will enable you to BLAST primers to map to parent sequence and also reverse compliment your reverse primer so you can match this to actual amplified sequence

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.basic.northwestern.edu/biotools/oligocalc.html

Note that the total amplicon size also includes the length of each primer region as well since the PCR extends (and joins) the nascent strand growth to each of the primer 3' ends. The final predominant blunt-end transcripts (amplicons) are thus inclusive of the primer lengths as well.  E.g., if Fwd primer is 20 nt, and the Rev primer is 20 nt, and the intervening sequence is 162 bp, the resulting amplicon product size is 202 bp.

Yes the total size of PCR product includes region of primers as well. I will send you detail steps if required..........by using primer 3 software. Just send me the raw sequences of Alkaline Phosphatase gene / or whether you wants to amplify total gene or a part of it. Also consider, are you going to do sequencing of part of gene or you wants to express this gene in some system by cloning......?

Oligo calc is for studying properties of primer. Its good software. For primer design I advise you to use primer 3 software..........

You can also utilize e-PCR which is part of the NCBI Resources:

http://www.ncbi.nlm.nih.gov/tools/epcr/

Once there, you can select the 'reverse e-PCR', put your primers in using the table input format available in the tabs, choose the dataset (e.g. Homo sapiens genome assembly, etc), select for exact matches or gaps, then 'Search'. 

Many thanks guys I did learn it

Hi Raha Osilan

You can get the size of your product in many methods, for example you can depend on your primer location and apply the equation as amplicon size=(location of reverse primer-forward primer)+1

and can applied by:

1. Inter NCBI and select your gene 

2. Obtain the sequence from Fsata

3. Click Ctrl+F and enter your primer (F), then you can complement the second primer (R) 

4. From this you can apply the equation above.

or

you can depend on the UCSC program and just enter your primers and the program will be calculate it.

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