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SP6 RNA Polymerase > FAQ |  | SP6 RNA Polymerase FAQ
See the Polymerases and Amplification FAQ also.
Q1: What is the molecular weight of SP6 RNA Polymerase?
Q2: What are the main causes of reaction failure using SP6 RNA Polymerase?
Q3: Is SP6 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
Q4: Does the reaction with SP6 RNA Polymerase require a primer?
Q5: What is the first base that SP6 RNA Polymerase transcribes?
Q6: Does SP6 RNA Polymerase leave an extra base at the end of a transcript?
Q7: Does SP6 RNA Polymerase require single stranded substrate?
Q8: Will SP6 RNA Polymerase work on single stranded substrate?
Q9: Will SP6 RNA Polymerase work on uncut plasmid DNA?
Q10: Can aberrant RNA be produced when using SP6 RNA Polymerase?
Q11: How can the yield of RNA be maximized when using SP6 RNA Polymerase?
Q12: Why is the specific activity of the probe low?
Q1: What is the molecular weight of SP6 RNA Polymerase?
A1: 98,532 Daltons (874 amino acids)
Q2: What are the main causes of reaction failure using SP6 RNA Polymerase?
A2: *Dithiothreitol is required for activity. Buffer older than 6 months should be supplemented with fresh DTT.
*SP6 is extremely sensitive to salt inhibition. Use drop dialysis or other purification to remove salt from DNA preparations.
*RNase contamination degraded the RNA product. Use RNase inhibitor at 1 unit/ml (provided in the HiScribe RNAi Transcription Kit (NEB# E2000). Use ultra pure water and make sure the DNA template has been phenol/chloroform extracted. Do not use a gel loading buffer containing SDS because it inactivates the RNase inhibitor.
Q3: Is SP6 RNA Polymerase an enzyme of choice for making high specific activity labeled probes?
A3: Yes, SP6 RNA Polymerase, T7 RNA Polymerase (NEB# M0251), and the HiScribe RNAi Transcription Kit (NEB# E2000) can be used to make RNA probes. Increased levels of detection in nucleic acid hybridization can be obtained due to the greater stability of RNA: DNA hybrids with respect to DNA: DNA hybrids (1). The NEBlot Kit (NEB# N1500) can be used for making high specific activity DNA probes using random primers. Klenow Fragment (3'→5' exo-) (NEB# M0212) can be used with the same procedure and a specific primer.
(1) Zinn, K et al. (1983) Cell 34, 865-879.
Q4: Does the reaction with SP6 RNA Polymerase require a primer?
A4: No, SP6 RNA Polymerase recognizes its promoter and starts transcription at the final G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template for 5’->3’ transcription.
Q5: What is the first base that SP6 RNA Polymerase transcribes?
A5: SP6 RNA Polymerase starts transcription at the final G in the promoter sequence. The polymerase then transcribes using the opposite strand as a template for 5’->3’ transcription. The first base in the transcript will be a G.
Q6: Does SP6 RNA Polymerase leave an extra base at the end of a transcript?
A6: Yes, like DNA polymerases that lack proofreading exonuclease activity, SP6 RNA Polymerase can add an extra base at the end of a transcript.
Q7: Does SP6 RNA Polymerase require single stranded substrate?
A7: No, SP6 RNA Polymerase displaces the DNA.
Q8: Will SP6 RNA Polymerase work on single stranded substrate?
A8: No, the promoter sequence must be double stranded.
Q9: Will SP6 RNA Polymerase work on uncut plasmid DNA?
A9: Yes, but SP6 RNA Polymerase is an extremely processive enzyme and will continue to transcribe around a circular template multiple times without disassociating. It is suggested that the plasmid be cut with a restriction enzyme that leaves a blunt or 5' overhang downstream of the DNA of interest.
Q10: Can aberrant RNA be produced when using SP6 RNA Polymerase?
A10: Aberrant RNA has been noted when the restriction enzyme used to linearize the plasmid downstream of the DNA of interest produces a 3' overhang (2). To avoid this, the plasmid should be cut with a restriction enzyme that leaves a blunt or 5' overhang downstream of the DNA of interest.
(2) Schenborn, E. T. and Mierendorf, R. C., Jr. (1985) Nucleic Acids Res 13, 6223-6236.
Q11: How can the yield of RNA be maximized when using SP6 RNA Polymerase?
A11: Higher yields of RNA may be obtained by raising NTP concentrations (up to 4 mM each). Mg2+ concentration should be raised to 4 mM above the total NTP concentration. Additionally, inorganic pyrophosphatase can be added to a final concentration of 4 units/ml to lower feedback inhibition. Note: NEB no longer sells inorganic pyrophosphatase (5/30/02).
Q12: Why is the specific activity of the probe low?
A12: The concentration of the radioactive nucleotide should be made limiting (12 μM). In other words, if labeled ATP is chosen, add 500 μM of UTP, CTP, and GTP but only 12 μM ATP. Thus every time an A is incorporated a higher percentage will be hot.
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