I have recently run into a few issues with my loxP constructs. Currently I have a floxed DSRed sequence with Stop codons functioning as a 'stutter' cassette for downstream genes. This way, my downstream genes are not expressed in the absences of Cre.However, we have found that we get 'leak', where the downstream genes are expressed even without Cre.
Common solution to this problem is to use two non-compatible Cre sequences around an inverted expression cassette (The FLEX system, described here: http://blog.addgene.org/plasmids-101-flex-vectors). This system seems ideal for my purposes, but my vector is a large, complex lentivirus and so I can't use any of the pre-designed AAV vectors so I will need to clone in this sequence.
The issue I am running into is that the standard FLEX sequence is 117 bp long. Ideally I want to order top and bottom oligos and just clone them in with restriction enzymes, but commercially oligos of over 100 bp aren't available.
Is it possible to simply shorten the FLEX sequence? Between the dual loxP sites there are spacers that don't appear to contain any relevant info. Can I simply shorten them?
For one, there are IDT Ultramers, custom DNA oligos up to 200 bases if you are willing to pay the price.
For another, I would think about cloning it by using shorter, overlapping oligos. You can generate two oligos of 80 bases, overlapping by 40 bases, to generate a whole sequence of 120 bp by primer extension. I used to use 5 µM oligos in a 20 µl HiFi polymerase reaction (Pfu / Phusion / Q5 / ...) and run a temperature protocol of 2 min each of 98°C / 30 / 40 / 50 / 60 °C followed by 10 min of 68 °C. I purify the poroduct and digest it to generate sticky ends.
Alternatively and more recently, I have repeatedly used NEBuilder (Gibson assembly) with bridging oligos. The way I do it is to have about 40 bp overlap (Tm > 55 °C) with the 5' ends binding to each other. This way it resembles a exonuclease pre-digested fragment that is normally generated as part of the Gibson assembly anyway. This is combine with the PCR-amplified, DpnI digested vector backbone (and potentially other fragments). Homology of 25 bases between oligo and vector backbone works well. Wither add backbone homology to your bridging oligos, add it oligo homology to your backbone PCR primers or split between the two as needed. I usually use 50 fmol of each fragment and 250 fmol of annealed oligos. Hope this helps.
Thank you so much! I had been leaning towards this strategy and its nice to hear someone else recommend it.
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