Question

Will having RNA affect the proper ligation of pAMP and pKAN?

My pAMP sample has a Nanodrop reading of 2.44 and my pKAN plasmid has a nanodrop reading of 1.9. I ligated the samples already and seemed to get accurate results as to what I was expecting and I just want to better understand how RNA can affect a ligation and the whole process of creating a cell with antiobiotic resistant genes. Please help.

Answer 1

Theoretically RNA can interfere less in the ligation but practically they don't effect significantly so there is no need to remove the RNA (which can be derived while extracting plasmid from bacteria. So, having RNA will not affect ligation.

For DNA sample if the ratio of 260/280 is between 1.8 to 2 then sample is having best DNA and for RNA it supposed to be 2-2.2 (plus minus 0.2), So we based on the nanodrop reading we can assume that pAMP is having some contamination of RNA (though some other factors can also affect the ratio like pH of the samples) and pKAN is having pure DNA as compared to pAMP.

As already mentioned above, RNA is not having any significant effect on the ligation process, similarly it will not alter ligation process while using antibiotic resistant genes like ampicillin (pAMP) and kanamycin (pKAN) and even numerous protocols does not have any concern regarding this..