I am using NEB mutagenesis protocol (not kit) for point mutation, primers are designed back to back orientation and after PCR I want to perform PNK phosphorylation. I have thermoscientific PNK enzyme with me and it says 5' termini of linear DNA (1to 20 Pmol) required for phosphorylation. 5' termini is confusing because I will take PCR product which has 5' as well as 3' end then what does 5' termini of DNA means?
I also want to know is heat inactivation of ligase required after ligation of phosphorylated product?
After ligation I will go for DpnI digestion so digested fragments will be present in the sample so presence of ligase enzyme could ligate it, that is why I want to know is heat inactivation of phosphorylated product is necessary?
Is 1 hr incubation for DpnI digestion ok?
Hi Monika,
Each PCR product has two strands of DNA. The "top strand" , when read from left to right, goes from 5' to 3' (by convention) and the bottom strand, when read in the same direction goes from 3' to 5', so both ends of the PCR product contain a 5' and 3'. The PNK only phosphorylates the 5' position of the nucleotide. Note that upon ligation the 5' end will ligate to the 3' end on the other side and the 3' to the 5', so each connection now has a phosphate group between them.
Concerning the inactivation of the ligase. I think it would be safe to assume that under normal circumstances the activity of Dpn1 will outcompete the activity of the ligase, so no need to inactivate the ligase.
The duration of Dpn1 digestion will be totally dependent on how much enzyme you add, in which volume and with how much PCR template. You would expect it to be fairly quick.
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