Hii everyone. I have a problem in case of pGEM T vector. I ligate my gene of interest and tag together into pGEM T vector but after transformation i get no colonies. I followed this protocol for ligation for 10 ul total volume
T4 Ligase 1 ul
10X buffer 1 ul
Insert DNA (56ng/ul) 7 ul
pGEM T vector 1 ul and keep O/N at 4 degree
What is the problem?
I also want to know how to calculate 1:3. 3:1 molar ratio of insert & vector?
Please show me with help of formula or calculation
How much 40 ng equal to how much micro liter?
Problem may be the gene of interest you eluted may be having impurities like salts preventing the ligation to occur. pGEMT-Easy vector is highly efficient so no problem with the vector. You can also check if T4 DNA ligase is working or not? Whether your competent cells ie., DH5 Alpha are working or not? To check that try to transform them with pET vector which is having kanamycin resistance and plate on Kanamycin selection plate. If you get colonies, your competent cells are working. When you dont get the results, you have to trouble shoot and suspect each and everychemical, vector to find out where it is going wrong. When i was not getting colonies, i changed the vector and tried at all the different concentrations of 1:1, 1:2, 1:3 of vector and insert. There is online calculator which calculates the amount of vector and insert you have to add . This depends on the size of the insert, size of the vector, concentration of the insert , vector and in what ratios you want to set up the ligationr eaction?
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