Multisite Gateway cloning using the Cloning Clipboard

With the introduction of the Cloning Clipboard in MacVector 12.7 both single step and Multisite Gateway cloning can be easily designed, replicated and documented.

The Cloning Clipboard allows you to assemble fragments by dragging compatible ends of digested fragments together to form new fragments. Every digest operation in a sequence window will place a fragment onto the Cloning Clipboard. To avoid confusion with digests that produce many fragments only single fragments from each digest operation are placed on the Cloning Clipboard. For example if you select two sites flanking a gene and digest it just the fragment from between the sites. Just as if you had cut out a fragment from a gel. The only exception is that digesting a single site from a circular sequence will place the linearized vector on the Cloning Clipboard.

The basic procedure is:

• Digest each fragment from each source vector until all required fragments are on the Cloning Clipboard.
• Assemble these on the Cloning Clipboard by dragging ends together.
• Insert this assembled fragment into the destination vector

Please note this tutorial assumes you want to take fragment A (in source vector A flanked by attL4 and attR1) with fragment B (in source vector B flanked by attL2 and attL1) and fragment C ( Vector C flanked by attR2 and attL3). These are to be placed in a destination vector with attR3 and attR4 (pDEST-R3-R4). However, the steps below are generic and apply to many multiway Gateway cloning procedures

Multiway Gateway cloning

1. Click PREFS, go to the MAP VIEW preference pane and click SET ENZYME FILE. Choose the Gateway Restriction Enzyme file and make sure USE ALL ENZYMES is selected.
2. Open the vector containing fragment A and select the attL4 and the attR1 sites flanking fragment A.



3. Select the DIGEST button
4. Open the vector containing the middle fragment (fragment B).



5. Select the attL2 & attL1 sites surrounding Fragment B and click DIGEST.
6. Now move to Vector C and select the attR2 and attL3 sites flanking fragment C. Again click DIGEST.



At this point the Cloning Clipboard will match the following screenshot



7. Switch to the Cloning Clipboard and select the attL1 site of Fragment B with the mouse button kept held down. Any compatible sites will be highlighted with a black “target”. Any incompatible sites will have gray “target”.
8. Drag this end to the attR1 site of fragment A. This should be the only compatible site.



The Ligation dialogue will appear. Both ends are compatible and shown as such by being highlighted in green. Click LIGATE



A new fragment will be created with these two fragments.

9. Now drag the attL2 site at the end of the new fragment towards the compatible site (attR2) on Fragment C



10. Select the final assembled fragment in the Cloning Clipboard
11. Open the Destination Vector file. In our example the two sites attR3 and attR4 will be highlighted in red and green to show that they are compatible with the selected fragment on the Cloning Clipboard
12. Select the fragment in the Cloning Clipboard



13. Drag the fragment from the Cloning Clipboard. A “+” will appear to show that the fragment has compatible ends.



14. Drop the fragment onto the vector.

    The ligation dialogue will again appear.

    

15. Ensure that the fragment is in the correct orientation and click LIGATE.



Note that the sites where the attR4/L4 and attR3/L3 have taken place are now attB4 and attP3.

A few points:

• All cloning steps are documented by the addition of a FRAG feature that includes the name of the construct, the enzymes/recombination sites and the date of the operation. By default these are hidden.



• Because the sequence of the att sites does change some sites contain ambiguities. Additionally the recognition sites used by MacVector are much shorter than the full sites. This is to ensure that sites are not missed, however, this does lead to false positives
• Please note that you will need to be using the upcoming MacVector 12.7 to do this.
• MacVector treats the recombination events used by the Gateway techniques as ligation events. The end result to the sequence is of course the same.

MacVector 12.7 will be released later this year. But you can download the updated Gateway file now.