problem in cloning work - (Apr/11/2005 )
hello,
is there any way we can check whether the ligation reaction is working or not, before we transform it into the competent cells?
because i have problems with transformation (my clone should be 18kb), and i dont really know which problem i should encounter, is it the ligation, or the transformation. as for now, i'm using E.coli DH5 alpha and JM109 competent cells, and both are not working with my ligation product, but can uptake my control plasmid (15kb) very well.
any suggestion? 
take a sample of your ligation mixture and run it on an agarose gel.
you should be able to see the insert and the vector, and various other bands of things joining up. hopefully there should be a band where insert+vector should be.
Or just try plating your transformation culture at a higher density. I recently just transformed using a 19kB plasmid and transformation efficiency suffers a lot at high plasmid sizes (using standard transformation protocols)>
you should be able to see the insert and the vector, and various other bands of things joining up. hopefully there should be a band where insert+vector should be.
i have actually ran the DNA sample on agarose gel, and some smear and light band have appeared on the gel. but how could we know that band is our actual ligation product, in other words, how could we confirm our ligation is successful?is it by comparing the band with the DNA ladder?
thanx for ur suggestion nway
you'd know it was successful by seeing a band where the product you've made should be. The only way you can really confirm that your ligation is working is by picking the colonies and screening them for your insert.
when my ligations weren't working,i ran a gel of the test subject, and the controls, you'd see the difference between the vector ligating back to itself, and the vector + insert playing nice. use a ladder.
if its smearing... do you clean up before ligation?
hope it helps.
when my ligations weren't working,i ran a gel of the test subject, and the controls, you'd see the difference between the vector ligating back to itself, and the vector + insert playing nice. use a ladder.
if its smearing... do you clean up before ligation?
hope it helps.
can i know what control you use for comparing those ligation product?
yes, i have purified my DNA before ligation. but it did smear anyway.
thanx for ur suggestion.
i guestimated. given that i knew the size of recircularised vector, i expected the vector+insert to be larger... and if the gods were smiling on me that day, there was a band in the right region.
i guess if you've got something the similar size as to what you're expecting, you could run that as a control.
the only time my ligation gel smeared was when the phosphotase nibbled at everything.
i guess if you've got something the similar size as to what you're expecting, you could run that as a control.
the only time my ligation gel smeared was when the phosphotase nibbled at everything.
i have repeated the ligation, this time around, when ran on agarose gel, they produced 2 obvious band, one band show about the same size of the vector, and another band was about the size of the insert.
i really cant tell whether they've ligated or not, because i was using lamda HindIII as my DNA marker, and between the highest size (which is 23 kb) and the second highest size (which is 9 kb), the range was too big. and the band of my ligation product was between that marker.
when i did the transformation (using CaCl2 method), no colony appear, but my control plate (which was the big size plasmid) was full of single colonies. the only other colonies that appeared was on my control plate- the ligation product on different selective antibiotic plate. my vector supposed to be kanamycin resistance, but the colonies that appeared were on LB-ampicillin plate. so i presumed that my ligation mixture was contaminated, for 3rd time in a row, or, there have been another resistance sites at my putative clone.
by the way, i've transformed in a higher density than before, still-no colony.
help me, i'm struggling with invisible thing here.
