picking primers - (Jan/19/2008 )

Hi,

I am trying to pick primers on primer 3 using the sequence which is shown below. Primer 3 is picking left and right primers but they start from somewhere in middle of the sequence. As I want to design primers for picking up this gene from cDNA library, I will have to use the primers which start from start codon(left primer) and stop codon (right primer). What changes do I need to make on primer3 to pick the primers for gene cloning? Also, is there any option on primer3 where you can insert restriction sites on the primers?

Thanks in advance

1 atgggagact cccacgtgga caccagctcc accgtgtccg aggcggtggc cgaagaagta
61 tctcttttca gcatgacgga catgattctg ttttcgctca tcgtgggtct cctaacctac
121 tggttcctct tcagaaagaa aaaagaagaa gtccccgagt tcaccaaaat tcagacattg
181 acctcctctg tcagagagag cagctttgtg gaaaagatga agaaaacggg gaggaacatc
241 atcgtgttct acggctccca gacggggact gcagaggagt ttgccaaccg cctgtccaag
301 gacgcccacc gctacgggat gcgaggcatg tcagcggacc ctgaggagta tgacctggcc
361 gacctgagca gcctgccaga gatcgacaac gccctggtgg ttttctgcat ggccacctac
421 ggtgagggag accccaccga caatgcccag gacttctacg actggctgca ggagacagac
481 gtggatctct ctggggtcaa gttcgcggtg tttggtcttg ggaacaagac ctacgagcac
541 ttcaatgcca tgggcaagta cgtggacaag cggctggagc agctcggcgc ccagcgcatc
601 tttgagctgg ggttgggcga cgacgatggg aacttggagg aggacttcat cacctggcga
661 gagcagttct ggccggccgt gtgtgaacac tttggggtgg aagccactgg cgaggagtcc
721 agcattcgcc agtacgagct tgtggtccac accgacatag atgcggccaa ggtgtacatg
781 ggggagatgg gccggctgaa gagctacgag aaccagaagc ccccctttga tgccaagaat
841 ccgttcctgg ctgcagtcac caccaaccgg aagctgaacc agggaaccga gcgccacctc
901 atgcacctgg aattggacat ctcggactcc aaaatcaggt atgaatctgg ggaccacgtg
961 gctgtgtacc cagccaacga ctctgctctc gtcaaccagc tgggcaaaat cctgggtgcc
1021 gacctggacg tcgtcatgtc cctgaacaac ctggatgagg agtccaacaa gaagcaccca
1081 ttcccgtgcc ctacgtccta ccgcacggcc ctcacctact acctggacat caccaacccg
1141 ccgcgtacca acgtgctgta cgagctggcg cagtacgcct cggagccctc ggagcaggag
1201 ctgctgcgca agatggcctc ctcctccggc gagggcaagg agctgtacct gagctgggtg
1261 gtggaggccc ggaggcacat cctggccatc ctgcaggact gcccgtccct gcggcccccc
1321 atcgaccacc tgtgtgagct gctgccgcgc ctgcaggccc gctactactc catcgcctca
1381 tcctccaagg tccaccccaa ctctgtgcac atctgtgcgg tggttgtgga gtacgagacc
1441 aaggccggcc gcatcaacaa gggcgtggcc accaactggc tgcgggccaa ggagcctgcc
1501 ggggagaacg gcggccgtgc gctggtgccc atgttcgtgc gcaagtccca gttccgcctg
1561 cccttcaagg ccaccacgcc tgtcatcatg gtgggccccg gcaccggggt ggcacccttc
1621 ataggcttca tccaggagcg ggcctggctg cgacagcagg gcaaggaggt gggggagacg
1681 ctgctgtact acggctgccg ccgctcggat gaggactacc tgtaccggga ggagctggcg
1741 cagttccaca gggacggtgc gctcacccag ctcaacgtgg ccttctcccg ggagcagtcc
1801 cacaaggtct acgtccagca cctgctaaag caagaccgag agcacctgtg gaagttgatc
1861 gaaggcggtg cccacatcta cgtctgtggg gatgcacgga acatggccag ggatgtgcag
1921 aacaccttct acgacatcgt ggctgagctc ggggccatgg agcacgcgca ggcggtggac
1981 tacatcaaga aactgatgac caagggccgc tactccctgg acgtgtggag ctag

---

If you need to amplify the whole sequence, then add restriction sites to the primer and then continue with 21 bases which will prime with the sequence. Have 6 bases As or Ts before the restriction sites for better digestion.

---

Hi Scolix,

Thank you very much for your reply.

You mean to amplify the whole sequence, I need not use the primer3?

Is there no option on primer3 to pick up primers for amplifying a whole sequence?

---

Honestly I donot really use primer3. I might use primer 3 if you wanted to find primers for sequencing. But to amplify the whole gene, its not necessary.

---

primer3 gives you the option to choose the sequence you want to amplify, haven't used it in a while but if i'm not mistaken you put the sequence between these simbols [CCGGTAGACGAATG....]

---