Life Ambion T7启动子转录试剂盒说明书

Appendix A Supplemental InformationAdditional proceduresA

Cap analog:

GTP ratio

0:1

1:1

2:1

4:1

8:1

10:1RNAyield 49.3µg43.5µg37.4µg25.7µg14.8µg12.0µgProtein yield (cpm)5,466cpm56,450cpm65,632cpm80,636cpm86,867cpm93,834cpm

T7-mMESSAGE mMACHINE reactions were done under standard conditions

except that the ratio of m7G(5')ppp(5')G to GTP was varied as indicated.

Reactions were incubated at 37°C using 1µg of T7 globin template DNA for 1hr. The globin RNA (6µg/mL) was then translated in a 25µL Retic Lysate IVT in vitro translation reactions for 60min at 30°C with 12.5µCi of [35S]methionine

(1200Ci/mmol). The incorporation of TCA-precipitable cpm was measured.

Optimizing yield of

short transcriptsThe mMESSAGE mMACHINE Kit is designed to function best with transcription templates in the 0.3–5kb range. Under these conditions, 1µg of plasmid DNA

template per 20µL reaction gives maximal RNA yield. Increasing the incubation time, template, or polymerase concentration does not generally increase the yield of the reaction. However, with smaller templates, these parameters may require adjustment to maximize reaction yields.

Several types of small transcript templates (<0.3kb) can be used in mMESSAGE

mMACHINE reactions. These include plasmid vectors containing small inserts, PCR products, and synthetic oligonucleotides which can either be entirely double-stranded or mostly single-stranded with a double-stranded promoter sequence (Milligan et al. 1987). Using oligonucleotides, and PCR-derived templates, almost all of the DNA is template sequence, compared to plasmid templates which include non-transcribed vector DNA.

1.Increase the reaction time

Increasing the incubation time is the easiest variable to change and should be

tried first. Try increasing the incubation time to 4or6hr. This allows each RNA polymerase molecule to engage in more initiation events.

2.Increase the template concentration

Increasing the template concentration is the next variable that should be tested. This can be helpful because, with short templates, the initiation step of the

transcription reaction is rate limiting. It is important to remember that 1µg of a short template contains a much larger molar amount of DNA than 1µg of a

longer template. 50ng of an 85bp PCR-derived template provides 0.9pmoles of template (and 0.9pmoles of promoters), compared to the approximately

0.3pmoles template in 1µg of the pTRI-Xef control template. In general, for

optimum yield of short transcripts, use about 0.5–2pmoles of template. For very short templates (i.e. ~20–30nt), use the upper end of this range.mMESSAGE mMACHINE® Kit User Guide21