Dear Researchers,
I have purchased my designed oligos from Sigma for miR based silencing studies as sense (~64nts) and antisense (~64nts); together if annealed it is ~64bp. I take equimolar concentrations of each stands to anneal (as far as mentioned in literature) followed by kinase treatment and ligation for cloning into a mammalian vector. I still then get negative clones and hence I doubt in taking the concentrations of oligos and I randomly tried 50,100 and 150 picomoles/ul for annealing. Could anyone suggest your comments in rectifying this?
Hello Snehaa,
I would suggest the following:
1. Check the concentration of your oligos, using the extinction coefficient provided by the supplier
2. Hybridization: I make a 20 µM solution of each oligo with some buffer (e.g. Tris-HCl 50 mM pH 8) plus 100 mM NaCl and I heat it in a beaker of boiling water, which I let cool to room temperature
3. Kinase treatment: There is no need to phosphorylate the hybrid. The 5' phosphates provided by the vector are sufficient. The ligation will yield a closed plasmid with staggered nicks, but they will be repaired in vivo. If you phosphorylate the oligo duplex, you will favour the formation of concatemers.
4. Cloning: make sure that the ratio between vector and insert is right (i.e. about 1:3 to 1:5) in terms of moles, not bulk weight. Adding a huge excess of your duplex is detrimental
Good luck
Pierre
Hello Snehaa,
I would suggest the following:
1. Check the concentration of your oligos, using the extinction coefficient provided by the supplier
2. Hybridization: I make a 20 µM solution of each oligo with some buffer (e.g. Tris-HCl 50 mM pH 8) plus 100 mM NaCl and I heat it in a beaker of boiling water, which I let cool to room temperature
3. Kinase treatment: There is no need to phosphorylate the hybrid. The 5' phosphates provided by the vector are sufficient. The ligation will yield a closed plasmid with staggered nicks, but they will be repaired in vivo. If you phosphorylate the oligo duplex, you will favour the formation of concatemers.
4. Cloning: make sure that the ratio between vector and insert is right (i.e. about 1:3 to 1:5) in terms of moles, not bulk weight. Adding a huge excess of your duplex is detrimental
Good luck
Pierre
Dear Pierre,
Thank you very much Sir. I follow the same protocol as you said for annealing as given in Sigma with 1x annealing buffer (10mMTris , 1mM EDTA and 50mM NaCl, pH 7.5 - 8) ; keeping it boiled for 5 mins at 95 deg and letting it cool down to RT for about an hour. Ligation, I prefer 1:3 (V:I) in terms of moles only. And I will check for kinase treatment for the vector (I use modified pcDNA3.1) and the extinction coefficient of the oligos with the datasheet once again.
Are you not using CIP to remove phosphate group from digested vector? You will not get negative clone(self ligated vector) if you treat your digested vector with alkaline phosphatase. Please ensure your ligase enzyme is working properly. Generally, Ligase requires ATP for its activity and ATP degrades very soon.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology