I am working on MTRR gene and i have specific primers from some article but i do not know what is the size that should i obtain ?
for a successful reaction , really i have PCR products and relatively all of them have the same size but i would like what is the specific size that should produce from those primers that i have ?

the 2nd question is how to calculate the ug that are needed for restriction digestion ?
please help me

Hello,
for your problem with the PCR product size I suggest you just take the reference sequence for that gene (you can find the complete sequence of all known genes for example on pages like www.ensembl.org; but make sure you choose the right species) and search for the primer sequence. When you found the forward and reverse primers you can just count. When I work on a new gene I always first copy the complete sequence into a word document and mark all my primers with the primer name as comment and save this as my personal reference. Thats also good if you later have to make additional primers. It keeps things organized.

For your restriction problem it would be good to know more about what you want to do with that and what kind of DNA (genomic, cDNA, plasmid) you work with, etc.

Good luck!

thank you sandra for your help bu i could not use www.ensembl.org so please if you can give me the hint to use it
and about the restriction problem i have genomis DNA and it is relatively low concentration
please give me the reply about how can i use the site

Hi,
to find your desired sequence in Ensembl you have to choose the species you work with and enter the gene name in 'Search Ensembl' on the start page.
You then get several results, usually the Ensembl and the Vega version of the protein coding gene; there can be differences in the number or size of the exons between the two; I usually choose the Ensembl version (habit).
You then get an overview of general information about the gene; to get the genomic sequence you click 'Genomic sequence' on the very left side.
On the following page you have to set the 'Markup options' (I'll tell you the ones I use):
-under '5'/3' flanking sequence' you can enter the length of sequence upstream and downstream of your gene that you want to have displayed
-for 'additional exons to display' I usually choose 'Ensembl exons' (habit)
-for 'orientation of additional exons' I choose 'display exons in both orientation'; though that's because that is the default setting
-for 'show variation' I choose 'yes'; all known polymorphisms will be marked up green in the sequence then. If you take 'yes and show links' you'll get a link for every polymorphism to another page with further information to it. But if you want to copy it to a text file it's better to leave the links out.
-for 'line numbering' I choose 'relative to coordinate system' or 'none'.
Then click 'Update' and you'll get the sequence below.

Considering the restriction: what species do you work with and what method is the restriction digest part of (for example cloning, AFLP, RFLP, Southern Blot)?

I hope this helps you a little; I'm not exactly an expert on this, I just know where to find what information I usually use.