TSAP thermosensitive alkaline phosphatase is a robust phosphatase that can efficiently dephosphorylate all 5´ termini from DNA fragments in 15 minutes at 37°C(1). TSAP is active in Promega restriction enzyme buffers as well as GoTaq® PCR Buffers(2). This allows TSAP to be used in simultaneous restriction digest/dephosphorylation reactions without the need for buffer exchange or serial reactions. TSAP is also irreversibly inactivated by incubating at 74°C for 15 minutes. Therefore, a DNA cleanup step is not required before proceeding to a ligation reaction. Here we examined whether TSAP can be used directly in reactions with NEB’s common restriction buffers.
The NEB restriction enzyme buffers chosen for this experiment include the standard NEBuffers 1, 2, 3 and 4, along with the unique buffers NEBuffer EcoRI and NEBuffer BamHI. According to NEB, the restriction enzyme EcoRI is 100% active in NEBuffers 1, 2, 3, 4 and the unique buffer supplied with the enzyme. Therefore, EcoRI was chosen to test these four common NEB buffers as well as NEBuffer EcoRI.
The Streamlined Restriction Digestion, Dephosphorylation, and Ligation protocol, provided with the TSAP Promega Product Information #9PIM991, was followed:
||15 units |
|10X restriction enzyme reaction buffer
|nuclease-free water to final volume
The digestion and dephosphorylation reaction was incubated for 15 minutes at 37°C, and the two enzymes were then inactivated at 74°C for 15 minutes. Since the NEB BamHI restriction enzyme is not inactivated by heat, the reaction was cleaned up using the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281). Following heat inactivation or cleanup, an aliquot of each reaction was religated using the LigaFast™ Rapid DNA Ligation System (Cat.# M8221):
|pGEM®-3Zf(+) Vector (cut, dephosphorylated and heat inactivated or cleaned up)
|T4 DNA Ligase
||6 units |
|2X Rapid Ligation Buffer
|nuclease-free water to final volume
The ligation reaction was incubated for 1 hour at room temperature, then the ligase was inactivated at 70°C for 10 minutes. Both unligated and ligated samples were analyzed side-by-side using 200ng vector DNA loaded on 0.8% agarose gels run for 1 hour at 50V.
TSAP activity was also tested in a functional cloning assay. Fifty nanograms of the unligated and ligated samples from the previous protocol were transformed directly into JM109 Competent Cells (Cat.# L2001) and plated on LB + 100µg/ml ampicillin plates overnight at 37°C. TSAP activity was assayed by counting the number of colony forming units (cfu) obtained from plasmid that had been digested, dephosphorylated and religated.
Figure 1, Panel A, shows the gel analysis of the 15-minute streamlined restriction digestion, dephosphorylation and ligation reactions using Promega restriction enzymes and Promega reaction buffers as a benchmark for TSAP activity. Lanes 3–6 show results in the presence of Promega EcoRI enzyme in Buffer H (with or without TSAP, with or without ligase) and lanes 7 and 8 show BamHI enzyme in Buffer E (with or without ligase). When TSAP was not present in the EcoRI digestion, the plasmid DNA can efficiently religate as shown by the high-molecular-weight banding pattern on the gel (Lane 4). When TSAP was added to the EcoRI and BamHI digestions then ligated, no high-molecular-weight bands were seen, confirming that TSAP was active in Promega reaction buffers H and E (lanes 6 and 8; reference 1). Figure 1, Panel B, demonstrates there is no difference in banding pattern between the 15-minute incubation and the extended 2-hour incubation.
Figure 1. Gel analysis of the Streamlined Restriction Digestion, Dephosphorylation and Ligation reaction using Promega restriction enzymes and digestion buffers.
pGEM®-3Zf(+) Vector was digested, dephosphorylated and ligated as indicated in the Methods section. The time of incubation for digestion and dephosphorylation was either 15 minutes (Panel A) or 2 hours (Panel B). Two hundred nanograms of each sample was run on an 0.8% agarose gel for 1 hour using 50 volts. Lane 1, BenchTop 1kb DNA Ladder (Cat.# G7541); lane 2, undigested pGEM®-3Zf(+) Vector; lanes 3–6, Promega EcoRI in Promega Buffer H; lanes 7 and 8, Promega BamHI in Promega Buffer E.
Figure 2 shows TSAP activity tested in NEB Buffers 1, 2, 3 and 4 as well as the unique buffers for the BamHI and EcoRI restriction enzymes (lanes 3–14). The plasmid DNA did not digest completely after 15 minutes when using the NEB EcoRI and BamHI enzymes under the same conditions as the Promega enzymes (see lanes 5–7 for the presence of uncut, supercoiled plasmid). The high-molecular-weight band observed may be the result of nicked or circular plasmid DNA that has not been digested to completion. However, the religated, high-molecular-weight banding pattern was not observed with any of the buffers, suggesting digested plasmid has been efficiently dephosphorylated by TSAP. Thus, TSAP appeared to be active in all NEB Buffers tested.
Figure 2. Gel Analysis of the Streamlined Restriction Digestion, Dephosphorylation and Ligation reaction using NEB restriction enzymes and NEBuffers.
pGEM®-3Zf(+) Vector was digested, dephosphorylated and ligated as indicated in the Methods section using NEB enzymes and reaction buffers. The time of incubation for digestion and dephosphorylation was either 15 minutes (Panel A) or 2 hours (Panel B). Two hundred nanograms of each sample was run on an 0.8% agarose gel for 1 hour using 50 volts. Lane 1, BenchTop 1kb DNA Ladder (Cat.# G7541); lane 2, undigested pGEM®-3Zf(+) Vector; lanes 3 and 4, NEB BamHI in NEBuffer BamHI; lanes 5 and 6, NEB EcoRI in NEBuffer EcoRI; lanes 7 and 8, NEB EcoRI in NEBuffer 1; lanes 9 and 10, NEB EcoRI in NEBuffer 2; lanes 11 and 12, NEB EcoRI in NEBuffer 3; lanes 13 and 14, NEB EcoRI in NEBuffer 4.
The restriction digestion and dephosphorylation incubation time was increased to 2 hours to completely digest the plasmid DNA in all buffers tested. The gel analysis of the 2-hour incubations showed the plasmid was completely digested after 2 hours in all buffers tested as all lanes have a single band (Figure 2, Panel B). In NEBuffers 2 and 4, smaller molecular weight bands were observed following the digest, possibly the result of star activity. For 2-hour digests, TSAP also appeared to be active in all NEB Buffers tested, determined by the absence of high-molecular-weight bands in the presence of ligase.
We wanted to also test TSAP dephosphorylation activity in a functional cloning assay. Even with plasmid DNA that looks completely digested by gel analysis, there can be trace amounts of uncut plasmid DNA, which will transform and contribute to colony formation. To account for this uncut background in the functional cloning assay, the colony forming units (cfu) from the ligated samples were compared to that of the unligated (Table 1). The control samples not treated with TSAP showed 30- to 50-fold increase in cfu after exposure to ligase, indicating successful ligation. No significant increase in cfu was observed with the ligated samples compared to the unligated samples if the samples were treated with TSAP, indicating successful dephosphorylation of the DNA by TSAP. There was an approximately tenfold increase in cfu in the unligated sample without TSAP. The results in Table 1 suggest that TSAP was active in all NEB Buffers tested compared to Promega Buffer H, which was used as the activity benchmark.
Table 1. Functional Analysis of the Streamlined Restriction Digestion, Dephosphorylation and Ligation Reaction.
Using the EcoRI restriction enzyme in Promega Buffer H as the benchmark, TSAP appears to be fully active in all NEB restriction enzyme buffers tested, which include NEBuffers 1, 2, 3 and 4, as well as the unique restriction enzyme buffers provided with the NEB EcoRI and BamHI enzymes. The 15-minute Streamlined Restriction Digestion, Dephosphorylation and Ligation protocol may be sufficient for use with Promega restriction enzymes and buffers; however, extended incubation times or gel purification are recommended to reduce background in cloning experiments.