I have similar colony numbers for my self-ligation (EcoRV + Cla1) and my re-ligation (EcoRv+BamH1+Cla1 with two inserts).
My No-ligation plate has no colonies at all.
I wonder whats wrong with my cloning.
I did Digestion at 37 degree
I did ligation at 16 degree overnight with ratio vector 1: inert 3:inert 3.
I did vector de-phosphorylation with SAP for 30 mins.
I did transformation using DH5α competent cells
First time I did digestion for 2 hours + SAP enzyme 0.5 in 50ul and I repeated the same protocol with digestion overnight + SAP enzyme 1ul.
Dear Maccus,
1. Before claiming that your cloning didn't succeed, did you check a set of clones for the presence of the properly inserted sequences (restriction pattern, colony PCR, sequencing) ? Even though it is likely that most or all of your clones contain no insert, just finding one correct one is all you need.
2. Since the ends generated by EcoRV and Cla1 are not compatible, why do you bother about vector dephosphorylation ?
3. If available, one way to counterselect empty clones is to digest the ligation mixture with a restriction enzyme whose site in the MCS lies between the two sites used for preparing the vector and at the same time is of course absent in the sequence of the insert.
Pierre Béguin I totally agree with your first opinion but my supervisor insist otherwise.
2. it is our lab's protocol to avoid any possibility of self-ligation. it doesn't hurt to do more anyway.
3. the empty clone is unlikely to happen because in my no-ligation plate (without adding ligase) none colony was found. Thus it means that there are certain degree of self ligation occurs in my self-ligation and plasmid ligation plates that appear to have certain amount of bacterial growth.
Because there is no self-ligation problem, there must be other limitations. You could increase the amount of insert and improve ligation conditions. Do you have a ligation control (plasmid digested with single enzyme and then ligated)? This will assure you that everything else is fine except the insert concentration.
Henry Daniell hi, no i do not have ligation control. I see your points in you suggestion, thats very interesting point of view. I am running a third run with another similar vector backbone. I saw some comments saying blunt ends ligate well at 4 degree Celsius, so I am now putting them in 4 dc ( around 6 hours) after 16hours at 16 dc until my transformation this evening. my insert ratio to vector is 3 to 1. I was thinking about that earlier as well, but I have seen many suggested that many people used 1:1:1:1 for 3 and more inserts. Only one lab member had done 3-piece insert with sticky ends (he suggested that maybe thats why I failed in blunt end ligation). I will see my cell transformation outcome tomorrow. Thank you so much for your suggestion.
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