Server at Davidson College
Paste or enter the single strand sequences of the plasmid and other components you are working with in FASTA format.
Enter the sequences in the 5' to 3' direction, with new sequences on separate lines.
The sequences will be screened for restriction enzyme sites.
Select from menu the restriction enzyme you will be using.
This will change the sticky ends that the generator uses as well as the cutting behavior of the enzyme.
Enter the desired melting temperature of the junction. Note that there is a minimum melting temperature, which will depend on junction sequence length. For junctions 18 base-pairs long, the minimum melting temperature is 44 °C. If you are having difficulty finding junctions, it is possible that your melting temperature is too low, which results in a large proportion of A and T in your sequence. Try raising melting temperature and re-submit. GGAJET uses the following equation to determine melting temperature:
Enter the desired length of the junctions in base pairs.
Number of desired junctions:
Enter the number of junctions you will need. Note that more junctions will require more run-time.
PCR Annealing temperature:
Enter the annealing temperature you will use, in degrees Celcius. Note that this field is optional.
If this field is left blank, GGAJET will assume an annealing temperature of 5 °C below the melting temperature.
Add one junction at a time and test against the plasmid. You can add a maximum of 10 junctions. Do not enter the reverse complement; it will be added automatically.
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