5. Remove the entire supernatant (~10ml) and sequence according to the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Protocol (79750)(3).
Sequencing of plasmids in E. coli colonies using the Thermo Sequenase Radiolabeled Terminator Cylce Sequencing Kit.
The rapid preparation of bacterial DNA from colonies described above, in combination with the Thermo Sequenase radiolabeled terminator kit, offers an excellent method to sequence plasmid DNA directly from colonies. Sequence information with virtually no artifacts or background is routinely generated and artifacts associated with template purity or premature termination are not seen at all. Gel compression artifacts, caused by G-C base-pairing of sequencing products, can also be eliminated using the dITP reaction mixture provided with the kit.
The results obtained using these protocols are comparable, or even superior to, those generated using template preparations of high quality and purity. It is important to note that the signal intensity of sequence is related to the size of the colony and the copy number of the plasmid. Therefore, colonies to be sequenced should be at least 2mm in diameter. If the size of the colony is smaller than 2mm or if plasmid is present in low copies, increasing the number of cycles should be beneficial. There is no difference in the quality of sequence generated from fresh or older colonies.
Note: The simple method of colony treatment before sequencing with the Thermo Sequenase radiolabeled terminator cycle sequencing kit was generously offered by Dr. R.S. Ranu at Colorado State University.
1. Life Science News , 20, Amershar