A synthetic cassette ready to be cloned without restriction enzyme digest should be designed so that the desired overhangs are part of the cassette. Match the overhangs to those generated by restriction digest of the vector you're cloning into.
If you find yourself a bit confused by 3' and 5' overhangs and what happens to the complementary strand, I'd suggest writing out the full sequence of the insert and the flanking restriction sites (including the complementary strand), and then making a line where the restriction enzymes would cut. Now you've bounded the sequence that you need to have synthesized for each strand. Use this 'drawing' to double check the sequences you have on the computer.
An example for cloning into and EcoRI and XhoI digested vector, in which insertion of the cassette restores the EcoRI site and destroys the XhoI site:
5' AATTCATATATATATATAG 3'
3' GTATATATATATATCAGCT 5'
- 22.5ul oligo at 10uM (pmol/ul)
- 6ul 5x T4 Ligase Buffer (yes ligase buffer, PNK buffer has no ATP!!!)
- 1.5ul T4 PNK (polynucleotide kinase)
Incubate at 37°C for 60 minutes.
1. Bring 800ml of water in a 1L beaker to boil. Remove from heat.
2. Combine full volume of each phosphorylated oligo into a single tube.
3. Place in floating rack in beaker of just-boiled water.
4. Allow to cool for approximately 90 minutes (this should work for any oligo cassette; the high and low temps required for any given cassette depend on length and sequence composition of the cassette).
Cassette is now at ~4pmol/ul, that's likely to be much more concentrated than any digested, gel-purified vector you have. So just a pinch will do....
In my experience the ligation efficiency with a well made cassette is much, much higher than even with the most carefully prepared insert made by digestion and gel-purification. Therefore, you can often get away with not gel-purifying your vector, and using a heat-inactivated sample as the backbone (of course the termini of the vector should still dephosphorylated). Although there may be a significant background from uncut vector when omitting gel-purification, the high efficiency of the cassette ligation will yield a substantial number of transformants above that background.
A mix for most of the time:
- 2ul 5x ligase buffer
- 0.7ul phosphorylated cassette (also try ~1/10 dilution of the cassette... too much can lead to multiple inserts)
- 7.0ul backbone
- 0.3ul Ligase
Incubate at RT for 60 minutes (for sticky ends), or at 16°C overnight (for blunt ends).
Transform 2ul into electrocompetent E.coli....