Designing Primers for PCR

[chesspuma99]

Dear Sir or Madam,

 

 

I do not really understand how to design a primer for a given DNA sequence. For example I have given this DNA sequence

 

841 aaaccagagc cggccagcca gtgaaacagc caccgtggag gggggacggc gaaaa***atg***aa

901 atccaaccaa gagcggagca acgaatgcct gcctcccaag aaacgtgaga tccccgccac

...

3241 catcgaaggc cgatctaacg tgggcaag***ta g***agaccttgc gggcagcgga ggcccggggc
3301 ctccttttac tgtctgtatc cagattactg tactgtaggc taagtaacac agtatttaca

 

 

Now I want to design Primers for a PCR to amplify the region marked by the start and stop codon. How do I do that? What do I need to concern? Can you recommand any literature where there is described step-by-step what to do in this situation? And how can I add sequences for EcoRI or BamHI.

 

I really need your help and I would be glad if someone could explain me the basic steps...

 

Best wishes,

chesspuma99

 

[Dagl1]

Dear Chesspuma,

In order to help you I need to know a few things; 
firstly, is this for PCR or qPCR as these things differ. In the case of PCR, you want to amplify this about 2300 bp fragment?

There is the NCBI tool "Primer blast" https://www.ncbi.nlm.nih.gov/tools/primer-blast/ which can give you an indication of primer combinations available.
I like this website; http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html for a more indepth look into primer design.
I can't remember right now, but there are other websites which you can use to verify primer specifications (also know that you should test your primer set once you obtain them, as computer simulations are not always right).

I am not entirely sure what you mean with "how can I add sequences for EcoR1 and Bamh1". Do you want to do digestions? 
Pasting the entire fasta sequence in here: http://nc2.neb.com/NEBcutter2/ will allow you to see enzyme cutting sites.

Hope that helps, 
Dagl1

[CharonY]

With regards to inserting digestions sites (or any sequences for that matter) you generally append them to your primer(s).

[Dagl1]

CharonY,

Could you explain what you mean by appending digestion sites to your primers?
I have done quite a decent amount of plasmid construction before, but I don't feel like I understand what you are referring to?

 

Kind regards,
Dagl

[hypervalent_iodine]

I believe CharonY means that you can include the sequence for the digestion sites in the primers, which will then mean that those sequences are amplified along with the rest of the sequence and integrated into your PCR products. 

[CharonY]

Exactly.

[Dagl1]

Ahh, I don't know why I didn't think of that. Thanks!