I want to clone a short DNA fragment of ~80 bp. The DNA fragment will be made by annealing two oligos together that were designed to generate 5' and 3' overhangs for the RE site, so the DNA fragment will "not be" digested with a RE. The plasmid will be digested, gel purified, and then treated with shrimp alkaline phosphatase followed by heat inactivation. Then the 80 bp DNA fragment (with the 5' and 3' overhangs for the RE site) will be ligated into the plasmid. Has anyone done something like this, and if so were there any issues, such as getting multiple DNA inserts?
Hi,
at my stage at Rouen University, we are doing the same, just with shorter inserts (30-50 bp). An issue you should be concerned about is the phosphorylation. You need to have your inserts or plasmid phosphorylated, but insert phosphorylation is better for sure. It is possible to order the phosphorylated oligonucleotides. Here, we use "cheaper" way and phosphorylate oligos with T4 polynucleotide kinase, just before annealing them together. Then, for the ligation, we use 12:1 (insert:plasmid) molar ratio. Sometimes we get double inserts, but usually in minority of clones. Hope this helps. Lucie
I did a fusion in a similar way with PCR and it worked fine. For such a construct, I had a 30-40 bp complimentary sequence to the plasmid. It might be difficult and you might need to try with different primer/DNA to plasmid ratios. Small inserts are sometimes difficult to ligate.
Hi,
at my stage at Rouen University, we are doing the same, just with shorter inserts (30-50 bp). An issue you should be concerned about is the phosphorylation. You need to have your inserts or plasmid phosphorylated, but insert phosphorylation is better for sure. It is possible to order the phosphorylated oligonucleotides. Here, we use "cheaper" way and phosphorylate oligos with T4 polynucleotide kinase, just before annealing them together. Then, for the ligation, we use 12:1 (insert:plasmid) molar ratio. Sometimes we get double inserts, but usually in minority of clones. Hope this helps. Lucie
I did this too even with a shorter oligo. I used a very high molar excess of the oligo (1:500, 10 kb vector and 40 bp oligo) and multiple insertion was not an issue. But of course, you should have a good strategy to check the single insertation.
For easyier screening of your clones i suggest you to insert a suitable RE site in the oligo sequence (if possible for your purposes).
I've done this frequently as well. If both sides of the insert have the same RE site then I always would dephosphorylate the vector and phosphorylate the insert. If I was doing directional cloning with different RE sites, then I would not dephosphorylate the vector and I would not phosphorylate the inserts. That way I could use vast insert excess and not have multiple inserts.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA