Hello,
I'm trying to clone oligos into my lentiviral vector. I've been having problems doing so, so I thought about optimizing every step of the process. Is there a way for me to check if the oligos annealed together before ligating them with the vector?
Hi Michelle, here's the way we check it:
Use 3 annealing reactions:
1) oligo sense only
2) oligo antisense only
3) oligo sense + antisense
Run a bit of each sample on a 2% agarose gel with ethidium bromide. Because EtBr binds with higher affinity to dsDNA then ssDNA, you should see a pretty intense band in combined oligo annealing if it worked, and only faint bands for the single oligos.
Best,
Gerrald
Hi Michelle,
I'm not sure about this but maybe it can work: plan you oligos so that they include a restriction site (ideally at the center of the oligos), then after annealing them try to digest them at that site. I think most restriction enzymes will only cut dsDNA, so if you run it on a very high percentage gel you should be able to see if you have shorter bands than those of the oligos themselves, which will mean you have had annealing.
I might try this myself now that I think of it, let me know if you happen to attempt it.
Ronni
You might be able to use a nanodrop. ssDNA has a higher absorbance by about 25% at room temperature. By comparing the absorbance of your annealed oligos to simply a mixture of the 2 should give you an idea how well they've annealed.
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/hyperchromicity
Good Luck,
Nathan
Hi Michelle, here's the way we check it:
Use 3 annealing reactions:
1) oligo sense only
2) oligo antisense only
3) oligo sense + antisense
Run a bit of each sample on a 2% agarose gel with ethidium bromide. Because EtBr binds with higher affinity to dsDNA then ssDNA, you should see a pretty intense band in combined oligo annealing if it worked, and only faint bands for the single oligos.
Best,
Gerrald
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