Dear All,
I am doing a ligation assited RCA. I have a padlock probe whose two ends are complementary with a target molecule in a contatenated way. I use HiFi Ligase to ligate the padlock probe. But I am struggling with the ligation efficiency and how to get the efficiency of the ligation (methods to test the ligation efficiency).
I did some literature research. In some papers, a PAGE electrophoresis is run over the unligated and ligated product(without any enzyme digestion). They claim if there is a band slightly lagging behind the unligated product in the lane of ligation product, your ligation is successful. Does anyone know the reason why we can make such a decision?
My concern is that not matter the ligation is successful or not, the band in the lane of ligation product is always lag behind that of the pure padlock lane as long as the target molecules keep binding on the padlock probe.
Best regards
Binbin Cui
Hi binbin,
- I am also working on RCA, for that, I also have to phosphorylate & ligate the DNA. I checked the ligation efficiency on 10% Denaturing PAGE. If ligation occurs then a thin band appears near the well whereas unligated DNA will migrate away from the well. You can easily compare the ligated DNA with unligated. For loading, use 300ng for unligated and 600ng for ligated DNA. Then You can go for Exo-digestion with ligated DNA.
Lane2- Unligated DNA
Lane3- Ligated DNA
Shrawan
Dear Shrawan Kumar ,
Is the PAGE running RCA product or ligation product?
I ran a 0.8% Agarose Gel electrophoresis with the RCA product. If I did the ligation step with 100pM target molecule and 200nM padlock probe, I could find a bright band in the well. But when I decrease the target molecule to 10 pM under the same condition, the band vanished. So I have no idea the low sensitivity resulting from the poor ligation efficiency or RCA efficiency.
BTW, I have a foundamental question. Will the target molecule dissociate with the ligated circle in the denaturing PAGE?
Binbin
Actually I used denaturing PAGE for checking the circular DNA, it is ligated or not. For RCA product, I ran on 0.5 agarose gel electrophoresis.
Hi Shrawan Kumar ，
If we directly run ligation product with denaturing PAGE, how can we know whether the bands above the unligated padlock probe is linear byproduct or circles we want ?
Bests
Binbin
Dear Binbin,
You can check the figure. The 3rd lane contain ligated DNA. A thin band is present near the well. In comparison I have also run the unligated DNA in lane. You can find the difference between them.
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