Hi I’m bit confused As I done ligation but I was trying to check if my ligation is successful or not I run my sample on gel but I didn’t found any band as I was having 6 sample .I’m trying to figure out the reason as all protocols for RD and ligation was accurate but on gel no band ? Anyone had this exp before or any other way to check successfully ligation.? Thanks
If I understand, the ligation mixture yielded no visible band when loaded on an agarose gel. Usually, when trying to analyze the products of a ligation reaction, one finds a set of bandcs that is not always simple to interpret: next to the properly ligated product, there are bands corresponding to unligated substrates and di- or oligomers of the vector or the insert. However, in your case, if you see no band, it is likely because the amount of these species is too low to be detected. Did you check (Nanodrop or equivalent) the amount of vector and insert that went into the reaction mixture ?
Pierre Béguin I was too confused but I done transformation and let’s see tomorrow result if there’s no colony next time I’ll check my transformation protocol at every step . And thanks for your answer I was also thinking same reason behind no bands cuz at least we need 20 ng DNA for visualization and as I just read some other people’s views also qpcr is optional.
Ligation is quite an ineffective process, meaning you will never get even close to 100% ligated product. Therefore, it may be hard to distinguish on a gel the ligated product next to the individual insert or vector (even if you have enough DNA to visualize, usually around 100ng).
As a general rule, it is not recommended to verify a ligation on an agarose gel. People mostly proceed with the transformation step right away and hope it will work. Successful transformation is the best indicator for successful ligation.
Did you transform the bacterial cells afterwards and does it work?
Some tips that might help you find the problem with ligation:
1. Success of ligation depends o the insert-to-vector molar ratio which is usually 3:1 or 5:1. You can find some of those calculators online, if you do not know how to calculate is yourself.
2. Which time and temperature do you use for incubation of ligation reaction?
3. I assume you use PCR products as inserts. You can analyze them on agarose gel prior to ligation to see how clean they are. The cleaner the bands, the better the ligation outcome.
Good luck!
Hello guys,
Running gel to check ligation efficiency is not a common practice but it does help in some circumstances (eg. contamination of ligase). I managed to visualize my ligated product and I attached a picture for your reference.
I set up a 1:3 vector: insert ratio with 100ng vector in 20ul reaction, and loaded 8ul to run the gel.
My insert and vector were similar in size that's why it appeared as a single band in the (-) lane.
Hope this helps!
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