PstI NEB#R0140

Protocol tips

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	Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates.

Protocol tips

Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates.

Publication protocol

Mitochondrial transcription elongation complexes were isolated essentially as described in Cheng and Gott (2000) with minor variations in dialysis conditions. Typically, mtTEC DNA was digested by incubating mtTECs (5 mg of protein) with 5–20 U of appropriate restriction enzyme in a final volume of 30 μL for 30 min at 30°C as follows: 20 U of HhaI (NEB), 5 or 20 U of AvaII (NEB), 20 U of NspI (NEB), 5 U of RcaI (Roche), 5 U of XhoI, 5 U of XhoII, 5 U of PstI (NEB), 20 U of MspI (Roche), and 10 U of SacII (NEB). Identical amounts of restriction enzyme were used in digestion reactions for both circle and hybrid cassette templates. Ligations were carried out for 30 min at 16°C with 500 μM ATP, 1.4 U of T4 DNA ligase (Roche), and 5 pmol of exogenous hybrid cassette DNA where appropriate. Run-on transcription reactions (35–50 μL) were similar to those described previously (Cheng and Gott 2000) except for slightly varying buffer conditions (due to individual restriction enzyme requirements); NaCl concentrations varied from 44 to 90 μM. Nucleotide triphosphates were used at 500 μM except as specified: CD1 (100 μM GTP), CD4 (100 μM UTP), HC1 (100 μM GTP), HC4 (100 μM UTP), CUp1 (100 μM GTP), CUp2 (100 μM UTP), and CUp3 (100 μM UTP)

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