In silico cloning - Bits.vib.be

This will allow you to choose which attB sites you wish to add to each end of the fragment. If you
have selected several fragments and wish to add different combinations of sites, you will have to
run this tool once for each combination.
Clicking Next allows you to extend the fragment with additional sequences by extending the primers
5’ of the template-specific part of the primer (between the template specific part and the attB
site). For example you can add a Shine-Dalgarno site between the attB and the gene of interest.
Press Shift – F1 and you will see a list of possible additions from which you can choose.
This list shows you the most common additions, but of course you can manually type or paste
whatever sequence that you want to add. Use the up and down arrow keys to select a tag and
press Enter. This will insert the selected sequence in the primer.
At the bottom of the dialog, you can see a preview of what the final PCR product looks like. In the
middle is the sequence of interest (i.e. the sequence you selected as input). In the beginning is the
attB1 site and at the end the attB2 site. The primer additions you have inserted are shown in color.
Note that the four terminal G’s are automatically added to the attB sites.
Clicking Next allows you to specify the length of the template-specific part of the primers.