Correction Notice: Expression and Purification of Recombinant Human Mitochondrial RNA Polymerase (POLRMT) and the Initiation Factors TFAM and TFB2M

After official publication in Bio-protocol (https://bio-protocol.org/en/bpdetail?id=4892&type=0), we found errors that should be corrected:

• • In the Recipes section under Part I: POLRMT purification buffers, the note under the 5% PEI solution should say “Adjust pH to 7.9…” instead of “7.0” to match the pH of all the other buffers.
• • In the Procedure section for Part I: POLRMT Purification, under part D. POLRMT AS precipitation, there is a math error that should be corrected to “______mL lysate × 0.351 g/mL…” instead of “3.51 g/mL”.
• • In step 11 of part E. POLRMT DEAE gravity column, The concentration speed should say “Concentrate POLRMT by centrifuging the Amicon filtration units at 1,300× g” instead of “1,900× g”. Having a lower speed will prevent protein precipitation issues and keep the centrifugation speed consistent with the later steps.
• • In section Part III: TFB2M purification, under G. TFB2M concentration, step 3 says “Start centrifuging the TFB2M sample at 2,500× g and gradually decrease the speed to 1,300× g if precipitate starts forming.” We have seen, however, that simply starting at a low speed of 1,300× g is better to prevent precipitation. The sentence should be changed to “Start centrifuging the TFB2M sample at 1,300× g.”