We are trying to ligate FokI digested insert with BsaI digested and rSAP treated Vector. We got 5-10 colonies in LB+ selected antibiotic plates after heat-shock transformation as well. But when we did colony PCR we got the bands similar to positive control i.e. undigested vector. I think it can not be due to self ligation of vector as the ends produced after digestion are incompatible and we have treated it with Shrimp-alkaline phosphatase to ensure that vector do not go self-ligation. So, We could not understand how we are getting colonies similar to vector and there is no insertion. Looking forward if any one has similar experience and how we could sort out this issue.
Not getting insert positive colonies even after rSAP treatment of the vector could mean
1. The digestion and purification of vector and insert were not efficient. (increase digestion time and use fresh buffers where the enzymes show their highest activity. Always gel cut purify the DNA to avoid undigested DNA contamination. Recheck if your insert primers have sufficient bases at the 5' end to allow for the enzyme to ocupy during digestion)
2. Molar ratio between the vector and insert was off. (try to keep the molar ratio around 1:5 or 1:7. Using higher ratios as well as lower ones retard efficient ligation)
3. Ligation buffer issues. (Ligation buffer needs to be fresh and free of preciptates. You can supplement 1mM ATP to the reaction if the buffer is old)
Sometimes, false positive clones are unavoidable. Therefore, always plate an Empty vector + Ligase - insert control to know how many false positives are present in the reaction. If the number of colonies in the Vector + Insert plate are the same or slightly more as the Vector only plate; do not screen it as the chances of getting a clone will be slim there. The difference between the plates is usually stark if everything went according to the plan. Change the ligation conditions and try again.
Praveesh Valissery Thank you so much these information. We tried optimizing digestion time and have used the buffer where enzymes have highest activity. we always conduct our ligation reaction in 1:1 and 1:3 ration we have not tried 1:5 and 1:7 vector: insert ratio. Let's see if it will give us positive result.
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