Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® KitTroubleshooting

a.Transcription of the control template is inhibited by the presence of your

template. (See Figure 3.A)

Figure 3Possible

outcomes of mixing

experiment

1 – control template

2 – experimental template

3 – mixture of 1 and 2A123B

123

This implies that inhibitors are present in your DNA template. Typical

inhibitors include residual SDS, salts, EDTA, and RNases. ProteinaseK

treatment frequently improves template quality.

Treat template DNA with ProteinaseK (100–200µg/mL) and SDS (0.5%) for

30min at 50°C, followed by phenol/chloroform extraction and ethanol

precipitation. Carry-over of SDS can be minimized by diluting the nucleic

acid severalfold before ethanol precipitation, and excess salts and EDTA can

be removed by vigorously rinsing nucleic acid pellets with cold 70% ethanol

before resuspension.

b.Adding your template to the reaction with the control template does not

inhibit synthesis of the control RNA. (See Figure 3.B)

This result indicates that the problem may be inherent to your template.

i. Use a different polymerase for transcription if possible.

Templates differ in transcription efficiency depending on the initiation

efficiency of their promoter, the presence of internal termination signals, and

their length. If the problem is due to the first or second of these issues,

changing the RNA polymerase promoter used to transcribe the fragment

may alleviate the problem.

ii. Check the amount and quality of template

Another possibility is that the template quantitation is inaccurate. If

quantitation was based on UV absorbance and the DNA prep had

substantial amounts of RNA or chromosomal DNA, the amount of template

DNA may be substantially less than the calculated value.

Also, check an aliquot of the template DNA on an agarose gel to make sure it

is intact and that it is the expected size.

iii. Extend the reaction time

Another parameter that can be adjusted is reaction time. Extending the

standard 1hr incubation to 4–6hr or even overnight may improve yield.mMESSAGE mMACHINE® Kit User Guide17