|
There are times when you might know the restriction map of a cloned fragment of DNA but you might not yet know the sequence. Such generic constructs can be created in GCK and the segments manipulated just like any piece of DNA whose sequence is known.
1. Start GCK. You will see an untitled Construct window as the active window. To create a generic construct of 5000 nucleotides, choose Construct > Insert Ns..., enter 5000 into the dialog and press OK.
2. You now have a linear construct containing 5000 Ns. Choose Construct > Display > Display Sequence to view the sequence as a series of Ns.
|
|
Figure 50: Place Sites Dialog
|
3. Choose Construct > Features > Place Sites.... You will see Figure 50. Select BamHI and EcoRI in the top list and press Add to move them to the bottom, Sites to Place, list.
|
|
Figure 51: Locate Sites Dialog
|
4. Select BamHI in the bottom list and press Locate to bring up Figure 51. Type in 200 in the Starting at position text box and press Add to add this location to the list of sites to place -- it will appear in the list of Instances at the right. Next type in the position 1000 and add it to the list, and then add 3456. You should have these three locations in the list on the right. Once you have done this, press Done.
5. Select EcoRI in the list (Figure 50) and press Locate. In the locate sites dialog that appears (Figure 51), enter 2000 and 4000. You have now defined the enzymes and their sites so press OK to actually place those sites.
|
|
Figure 52: Placed Site (Sequence View)
|
6. In Figure 52 you can see that the BamHI recognition sequence has actually replaced some of the Ns. This is therefore a 'real' site that can be cut with the enzyme and used to produce fragments from this DNA.
|
|
Figure 53: Placed Sites (Graphic View)
|
7. Choose Construct > Display > Display Graphics to bring up Figure 53. You can now select fragments and use them in cloning steps as was done for fully sequenced DNAs in previous tutorials. As more sequence information becomes known for this construct, you can use the real sequence to replace the Ns in this generic construct.
|
