Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® KitTroubleshooting

Total cpmx100=% incorporation

Calculation of yield

Once the percent incorporation of radiolabel is known, it can be used to calculate the mass yield of RNA transcribed in the mMESSAGE mMACHINE® reaction. The

concentration of GTP limits the amount of RNA that can be synthesized. For any tracer other than labeled GTP (e.g. [α-32P] UTP), each 1% incorporation corresponds to about 2µg of RNA synthesized in a T7 or T3 reaction. For SP6 Kits, each 1% incorporation corresponds to about 1.3µg of RNA synthesized.

1% incorporation = 2µg/RNA

For example: if % incorp. = 10%

10 x 2µg = 20µg RNA

In a T7 or T3 reaction, if all four nucleotides are incorporated equally, 39.6µg of RNA will be produced if all of the 1.5mM of GTP is incorporated into RNA (the sum of the molecular masses of the 4nucleotides in RNA is about 1320). Since the ratio of cap analog to GTP is 4:1, this represents a maximal theoretical incorporation of 20% of the label.

1.5mM 1320gx10µL1000mMx20µL39.6x103g=10=39.6x10–6g = 39.6µg

The standard SP6 mMESSAGE mMACHINE® reaction contains 1.0mM GTP, so when that value is substituted in the above equation the maximum theoretical yield for SP6 mMESSAGE mMACHINE® reactions is 26.4µg.

Troubleshooting

Use of the Control

TemplateThe pTRI-Xef control template is a linearized TRIPLEscript plasmid containing the 1.85kb Xenopus elongation factor 1α gene under the transcriptional control of tandem

SP6, T7, and T3 promoters (pTRI-Xef1). Any one of the three RNA polymerases can be used to synthesize the control RNA. When transcribed with the following RNA polymerases, sense transcripts of the indicated length are produced from this

template: Enzyme

SP6

T7

T3Transcript Size1.92kb1.89kb1.86kb

These transcripts will produce a 50.2kD protein when translated.

1.Reaction setup

Use 2µL (1µg) of pTRI-Xef in a standard mMESSAGE mMACHINE® reaction as described in section“Capped transcription reaction assembly” on page 11.

mMESSAGE mMACHINE® Kit User Guide15