2 replies to this topic

[Fluffy]

Hi Everyone,
I have to design forward and reverse primers for the full sequence of my two genes (ubiquitin conjugating enzyme genes). Primers have to include 5' restriction enzymes. My restriction enzymes are NdeI (forward primer) and XhoI (reverse primer). I was told that since I am designing primers to amplify full sequence of each gene, the start and stop codons in each gene sequence have to be considered. Primer length is 18-25 ntds preferably, GC content below 50%, and Tm range is 58-60C. I am not good at using softwares for primer design, so I would appreciate the help of anyone able to use primer design tools. The gene sequence for my two genes are as follows:

Gene 1 sequence ID 388206
1 ctcttcttcc atttctttca aaattaaagt attgttactc tgctattggc tcaaaacctc
       61 tgcaatctcc gtctccttca atttcaactc aagcaaatcc acctctttca ctagtttcat
      121 cactttcaga tcagggtttg gagttgaagg tacggggggc taattgatgg cgtcgaagag
      181 gatattgaag gagctcaagg atctgcagaa ggatcccccc acatcatgca gtgctggtcc
      241 agtggcagag gatatgttcc attggcaagc aacaatcatg gggcctaccg atagccctta
      301 tgctggaggt gtatttttgg tttcaattca tttccctcca gattatcctt ttaagcctcc
      361 aaaggttgcc ttcagaacta aggttttcca tcccaacatc aacagcaatg gaagtatttg
      421 tctggatatt cttaaggagc agtggagtcc agcattaacc atatccaagg tcctgctgtc
      481 catctgctct ctgttgacag acccaaaccc agatgatcct cttgtacctg aaattgctca
      541 catgtacaag actgacaggg ccaaatacga aaccactgct cgtagctgga ctcagaaata
      601 tgcaatggga tgatgcgcaa aatgtctcca ggcatgtctg ggactttgta acagcaatgt
      661 cttatgtgct tggggtgaat gaataaattc cgtgaaagaa cttagttact tcttaatctc
      721 ccttcatgag ggttgttaag ggaacagctg ttttcaattt gtgaatattt atttgatgac
      781 tagtaaggga gaaactgcaa tgtaattcta ctttgtttgc cagtt

Gene 2 Sequence ID 886678
        1 gcacgagggc gacttttgca taaaccaaaa ttagaatcaa attggaagag agaaaaaaaa
       61 tggtggactt ggctagggtt caaaaggagc tccatgaatg caacagagat gttcaggttt
      121 ctggaattaa tgttaccctt aaaggtgaca gtctcactca cttgattggt acaatccctg
      181 gtcctgttgg tactccttac gaaggcggta ctttcaagat cgatatcact cttactgatg
      241 gctacccatt tgagcctcca aaaatgaaat tcgccacaaa agtttggcat cccaacataa
      301 gtagtcaaag tggagcaata tgcctagaca tcctgaagga ccagtggagc ccagcactaa
      361 ctctcaagac agctctcctt tctatacaag cattactttc tgctcctgaa cctgatgatc
      421 cacaagatgc agttgttgca cagcagtatc ttagagaaca tcagaccttt gtcggcacag
      481 ctcgttactg gactgagact tttgcaaaaa catccacact tgctgcagac gacaagatac
      541 aaaagcttgt ggaaatgggc tttcctgaag ctcaagtgag gagtactttg gaagcaaatg
      601 gttgggatga aaacatggct cttgaaaagc tgttgtccag ctaaaaccct tctactgcaa
      661 ctcatatttt gataagacaa ttatatcctt ccagcaaaag ctgatgacta gaatagagtc
      721 actcggttat actgttgctt ggcaatcttg tttctgtctc ctttatggtt tgctgttgac
      781 atctcttcat atcctgtgaa gattctgatg ttatttttac aatatcaagc aaattgcata
      841 tgaatcatgg ggaggaagtg gactttccgg ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa
      901 aaaaaaaaaa aaaaaaaaaa aaa

Thanks Everyone :D

[bob1]

First of all you need to identify where the start and stop codons are in those sequences... unless you want to clone the UTRs also.

If you want to clone the full sequence excluding UTRs, then you have no choice about where to place the primers, other than length... so your specific primers are the start 18-25 bp and the final 18-25 bp (reverse complemented) of your gene sequences.   YOu can calculate the Tm by using the following formula: Tm= 4*(number of G and C) + 2*(number of A and T).  You can adjust the length of the primers to compensate for differences in the sequence giving different annealing temperatures.

The annealing temperature you will use in the PCR is calculated based on the specific component only, ignoring the bits added as these don't play a part in the PCR initially.

Restriction sites and other add-ons go on the 5' end of the primer always.

You will probably want to add a kozak sequence (GCCACCATG, where the ATG is the start codon of your gene) to the 5' end if you are planning on expressing these in eukaryotic cells.

So your forward primer will look something like:
[Nde1site][kozaksequence][18-20bp of codingsequence]

Reverse will look like:
[Xho1site][reversecomplement of 18-20 bp of codingsequence]

[Fluffy]

Hey Bob1
Thanks for your help!
Greatly appreciated