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?
There are several potential issues.
First, are there restriction site overhangs after your oligos are annealed? I notice that your two 64 bp constructs generate an annealed product of 64 bp, but it should be 70-72 bp. For example, if your restriction sites are BamHI and EcoRI, after annealing your oligo should look like this:
Top - GATCCxxxxoligoxxxG
Btm - GxxxoligoxxxxCTTAA
This way, after annealing your oligo will be able to directly anneal into your digested vector. If not, you will need to digest your oligo before ligation into your vector.
As far as concentration goes, I have had great success when diluting my intial oligo stocks to 100 uM (i.e. multiply the nmol supplied by the company by 10 and add that many uL of dH2O). I then add 1 uL of each top and bottom stock into a 10 uL reaction with ligase buffer and PNK. After that, dilute 1:100 (100nM/uL final) and ligate approx 1 uL of annealed oligo into 50 ng of vector (assuming vector
Dear Levi,
Thank you for your valuable suggestion. My insert oligo contains the overhangs of miR155 on either sides. It is actually designed in such a way that it has the miR155 backbone with modified sense and antisense regions containing siRNA sequences of targets. Therefore, its only 64bp after annealing. I did tried treating the top and bottom oligos with kinase separately (20ul reaction) followed by annealing using 1x annealing buffer and that was found to be failed at last. I will try with your suggested protocols this time :)
Most likely your problem is not in the oligos but in the quality of the linearized vector prep you are trying to ligate into. Did you check un-ligated vector to make sure that you don't have any circular molecules still present? Also, how efficiently is your vector phosphatased? Self-ligation is much more efficient than intermolecular ligation.
Dear Mark Farman,
I phosphatase only oligos and not the vector. The partial restriction digestion for the vector is already optimized. Yes the possibility of self ligation is highly possible than the other as you say.. Can u tell me how to check if the vector is phosphatased so that I can try it as well. And moreover, as the size of the oligos is small and is same as sense and anti sense, I couldn't confirm them after annealing followed by Kinase by running an agarose or by nanaospec. As I couldn't find the conc. of oligos quantified by nanospec in any of the papers I read, I m stucked off in this step. Thank you very much for your suggestion!!
Snehaa, you kinased your oligos to ADD phosphates. Phosphatasing of the vector REMOVES the terminal phosphates, thus preventing self-ligation. Your answer tells me two things: if you used partial digestion to generate your vector, then you definitely have undigested molecules remaining in your prep. Second, if you did not phosphatase the vector, the linear fragments are self-ligating.
Note the key is to show that ligation in absence of insert gives very few colonies while inclusion of insert gives many. Then you can be sure that most picked colonies contain insert.
Dear Mark Farman,
Thank you so much! I m clear now to give my next attempt :)
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