mMESSAGE mMACHINE™ T7 Transcription Kit - FAQs

If the RNA appears degraded (e.g. smeared), remove residual RNase from the DNA template preparation before in vitro transcription. Do this by digesting the DNA prep with proteinase K (100-200 µg/mL) in the presence of 0.5% SDS for30 min at 50°C, follow this with phenol/chloroform extraction. The RNase Inhibitor that is present in the transcription reaction, can only inactivate trace RNase contamination. Large amounts of RNase contamination will compromise the size and amount of transcription products.

The pTri-Xef control template that comes with mMESSAGE mMACHINE T7 Transcription Kit (Cat. No. AM1344) is a linearized TRIPLEscript DNA plasmid containing the 1.85 kb Xenopus elongation factor 1α gene under the transcriptional control of tandem SP6, T7, and T3 promoters (pTRI-Xef 1). While we cannot provide a full map of the linearized plasmid, the insert sequence with the three promotors is as follows:

ACGATTTAGGTGACACTATAGAATACACGGAATTAATACGACTCACTATAGGGAGACTCGAGAATTAACCCTCACTAAAGGGAGGTACCGCGGATGCATGAATTCCGCCGGTGAGTTCTAACTATCCACCGCCAAACATCTAATCCACAATGGGAAAGGAAAAGACTCACATCAACATCGTTGTCATTGGACACGTAGATTCTGGAAAGTCCACAACAACTGGACATCTGATCTACAAATGTGGTGGTATCGACAAGAGAACCATCGAAAAGTTCGAGAAGGAAGCTGCTGAGATGGGAAAAGGCTCCTTCAAGTATGCCTGGGTCTTGGACAAACTGAAGGCCGAGCGTGAACGTGGTATTACCATTGACATCTCCCTGTGGAAATTTGAGACCAGCAAATACTATGTCACTATCATTGATGCTCCTGGACACAGAGATTTCATCAAGAACATGATCACTGGTACTTCTCAGGCTGACTGTGCTGTCCTGATTGTTGCTGCTGGTGTTGGTGAATTTGAAGCTGGTATCTCAAAGAATGGACAAACCCGTGAGCATGCCCTCCTTGCCTACACTCTGGGTGTAAAACAGCTGATCGTTGGTATTAACAAGATGGATTCCACTGAACCCCCATACAGTCAGAAAAGATATGAGGAAATTGTAAAGGAAGTCAGCACATACATCAAGAAGATTGGTTACAACCCTGACACTGTTGCCTTTGTACCTATTTCTGGATGGAACGGTGACAACATGCTTGAGCCTAGCCCCAATATGCCATGGTTTAAGGGATGGAAAATCACCCGTAAAGAAGGATCTGGCAGCGGAACTACCCTGCTGGAAGCTCTTGACTGCATTCTGCCACCTAGTCGCCCAACTGATAAGCCTCTCCGTCTGCCTCTGCAGGATGTCTACAAAATTGGCGGTATTGGTACTGTACCAGTTGGTCGTGTGGAAACTGGTGTCATTAAACCAGGCATGGTGGTTACCTTTGCCCCTGTTAATGTAACAACTGAAGTGAAATCTGTTGAAATGCACCATGAAGCCCTTACTGAGGCCGTGCCCGGTGACAACGTCGGCTTTAACGTCAAGAACGTTTCCGTGAAGGACGTCCGTCGTGGCAACGTTGCTGGTGACAGCAAGATTGACCCACCAATGGAAGCTGGTTCTTTTACCGCACAGGTTATCATCCTGAATCACCCAGGCCAGATTGGTGCTGGATATGCCCCTGTGTTGGATTGCCACACTGCTCACATTGCTTGCAAGTTTGCTGAGCTAAAGGAGAAGATTGATCGCCGTTCTGGTAAGAAACTGGAAGACAATCCCAAGTTCCTGAAGTCTGGAGATGCTGCCATTGTTGACATGATCCCAGGAAAGCCAATGTGCGTGGAGAGCTTCTCAGACTACCCTCCTCTTGGTCGTTTTGCTGTCCGTGACATGAGGCAGACTGTTGCCCTAGGAGTCATCAAGGCAGTGGAAAAGAAGGCAGCAGGATCTGGCAAAGTCACAAAGTCTGCTCAGAAAGCAGCGAAAACCAAATGAATATTTCCAGAGTCTCTCACCTCAGTCATAACCAGTGGTGGAGGATTGGTCTCAGAACTCTTTCTATCAATTGGCCATCAGAGTTTAATAGTAAAAGACTGGTTAATGATTACAATGCATCGCAAGAGCTTCAGAAGGAAAAAAATGCTTGTGGACACATTTGTTTGTGGCAGTTTTAAGTTATTAGTGTTTTAAATCCAGTAATTTCTAAATGGAAGCAAATTGACCAAAATCTGTCACCAAATTTGAGACCATTAAAAAAAAGTTGAAAAGATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGGAATTTCGCGGAATTCGAGCTCGCCCGGGGATCCTCTAGAGTCGACCTGCAGCCCA

SP6 (ATTTAGGTGACACTATAGA) 1.92 kb
T7 (TAATACGACTCACTATAGG) 1.89 kb
T3 (AATTAACCCTCACTAAAGG) 1.86 kb

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