The 3'-5' exonuclease activity of the large fragment of DNA polymerase I (Klenow enzyme) was utilized to convert the 3' sticky ends into flat ends. The method is to add the Klenoow fragment (final concentration 5U/ug) to the in vitro transcription system before the addition of nucleotides and RNA polymerase, incubate at 22℃ for 15mim, and then add the nucleotide mixture and RNA polymerase.
Operation method
RNA standard transcription reaction
Materials and Instruments
Template DNA or plasmid Move I Materials and equipment Caveat 1) Low RNA recovery when using the standard system. Possible reasons include: ① Insufficient amount of DNA template in the transcription system Since the DMA template can be precipitated by spermidine in the transcription buffer, be careful to mix the components at room temperature and pay attention to the order of sample addition. High NaCl concentration (>30 mmol/L). High concentration of NaCl (more than 30 mmol/L) will seriously reduce the activity of RNA polymerase (only 50%), so the salt should be removed by column chromatography and washed with 70% ethanol for 1~2 times. RNAase contamination. RNAase inhibitors (e.g., RNasin) must be added to all in vitro transcription systems, and all solutions should be prepared in the kit as much as possible, and 0.2% DEPC water should be used as the solvent for self-prepared solutions. RNA polymerase inactivation: RNA polymerase activity can be determined by jffi synthesizing a set of positive controls.2) RNA transcripts are incomplete due to the following reasons: ① RNA synthesis is terminated prematurely. Add cold rNTP (the same kind as isotope-labeled rNTP but without isotope labeling) to the system to increase the efficiency of full-length transcript synthesis. In addition, the reaction temperature can be lowered from 37°C to 30°C. ② Presence of polymerase terminator sequences Re-subcloning of the inserted fragment into another vector with a different promoter, some sequences can be recognized as terminator sequences by one RNA polymerase, but not by another RNA polymerase. ③ RNA sample buffer degradation. If the RNA sample buffer is too long or repeatedly frozen and thawed, the RNA may get a migration distance that does not match its true size.(3) A possible reason why the RNA obtained is longer than expected is that the transcription was initiated from the wrong strand of DNA. If the endonuclease used to linearize the DNA template produces 3' sticky ends, the resulting RNA may be longer than expected, and if the use of such restriction endonucleases cannot be avoided, the DNA should be flattened by Klenow blockbuster treatment. For more product details, please visit Aladdin Scientific website.
TE Isopropanol 3mol LNaAc Anhydrous Ethanol 5× Transcription Buffer l00 mmol L DTT RNasin rATP rGTP rUTP 2.5 mmol L each [α42P] rCTP SPG T3 or T7 RNA Polymerase TE-Saturated Phenol Chloroform Isoamyl Alcohol DNase 7.5mol L Ammonium Acetate Anhydrous Ethanol Ethanol 0.5mol LNa2 HPO4 Agarose
DE81 membrane Liquid flash meter Electrophoresis tank Centrifuge
1) Restriction endonuclease
2) Template DNA or plasmid
3) TE
4) TE saturated phenol: chloroform
5) Chloroform: Isopropanol (24:1)
6) 3mol/LNaAc (pH 5.2)
7) Anhydrous ethanol
8) 5×transcription buffer: 200 mmol/LTris-HCl(pH7.9), 300 mmol/LMgCl2, 10 mmol/L spermidine, 50 mmOl/LNaCl
9) l00 mmol/LDTT
10)RNasin
11)rATP, rGTP, rUTP, 2.5 mmol/L each
12) [ α42P ]rCTP(50uCi,10uCi/ul)(lCi=3.7X1010Bq)
13)SPG,T3 or T7 RNA polymerase
14)TE-saturated phenol:chloroform:isoamyl alcohol (25:24:l, pH 4.5)
15)DNase
16)7.5mol/L ammonium acetate
17) anhydrous ethanol
18)70% ethanol
19) DE81 Membrane
20)0. 5moJ/LNa2HPO4( pH7,0)
21) Liquid flash meter
22) Agarose
23) Electrophoresis tank
24) Centrifuge
II. Methods of operation
1. Preparation of DNA template
1) Determine the restriction sites within or downstream of the cloning site that will produce the desired run-offtranscript. Whenever possible, select restriction enzymes that produce 5' protrusions or sticky ends. If the enzyme used produces a 3' protruding end, it can be replaced with another enzyme or flattened.
2) Check the completion of the digestion by agarose gel electrophoresis. At this time, put the DNA sample on ice, if the digestion is complete, then continue to the next step, otherwise, add restriction endonuclease to the DNA, react for 30 min, and then analyze by electrophoresis. Be careful not to add more than 10% of the total volume of the enzyme, as the enzyme is stored in glycerol, and too much will affect the enzyme activity.
3) Add equal volume of TE saturated phenol; chloroform, mix and spin for 1 min to extract DNA, centrifuge at 12000 g for 2 min, transfer the upper liquid phase to a new tube, add 1 times the volume of chloroform: isopropanol (24:1) mix and spin for lmin, centrifuge at 12000 g for 2 min.
4) Transfer the upper liquid phase to a new tube, add 0.1 times the volume of 3 mol/L NaAc (PH5.2), 2 times the volume of anhydrous ethanol to precipitate DNA, place at -70℃ for 30 min, centrifuge at 12000 g for 2 min.
5) Carefully pour off the supernatant, precipitate with 1 ml of 70% ethanol, centrifuge at 12000 g for 2 min, discard the supernatant, dry the precipitate under vacuum, and resuspend the DNA samples in sterile water or TE buffer to a concentration of about lmg/ml.
2. In vitro transcription reaction
In vitro transcription systems generally require the addition of isotopic labeling to enhance the sensitivity of the assay. rNA transcription can be labeled with 32P-, 33P-35S-, or 1 H-labeled ribonucleotides, and of the four types of ribonucleotides, only rCTP and rUTP are used for isotopic labeling. The specificity of commonly used labeled nucleotides is given in Table 3.2.
The following is an example of an in vitro transcription system using " α32P " rCTP and illustrates the procedure as follows.
1) Add in sequence:
5X transcription buffer 4ul
DTT, 100 mmol/L 2ul
RNasin 20-40U
rATP,rGTP,rUTP(2.5 mmnol/L each) 4ul
Linear template DNA (0.2~1 mg/ml) 1ul
(a-32P)rCTP(50uCi, 10uCi/ul) 5ul
SP6, T3 or T7 RNA polymerase 15~20U
Add water without RNase to a final volume of 20ul.
The above components should be mixed at room temperature. If handled, DNA will be precipitated by spermidine in the buffer.
2) React at 37~40℃ for lh, and then add RNase-free water to the final volume of 20ul.
3) Remove 1ul to determine the isotope incorporation efficiency. The remaining sample can be digested with RNase-free DNase.
3. Purification of transcribed RNA products
After the in vitro transcription reaction, the DNA template can be removed by DNAase I. This results in a highly sensitive RNA probe. The removal of the DNA template is also required for biologically active RNA samples, where it is critical to maintain the integrity of the RNA. DNase is an RNAase-free DNA enzyme that effectively removes the DNA while maintaining the integrity of the newly synthesized RNA.
1) Add DNase to the in vitro transcription system at a final concentration of 1U/ug of template DNA.
2) Incubate at 37℃ for 15 min.
3) Extract the protein with TE-saturated phenol:chloroform:isoamyl alcohol (25:24:l, pH 4.5). Vortex for 1 min, centrifuge at 12000 g.
4) Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), vortex for lmin, centrifuge at 12,000 g.
5) Transfer the upper aqueous phase to a new centrifuge tube, a small amount can be analyzed by denaturing gel electrophoresis, and the leftover can be further precipitated and recovered.
4. Precipitation and recovery of the transcribed RNA product
1) Add 0.5 times the volume of 7.5mol/L ammonium acetate. 2) Add 0.5 times the volume of ammonium acetate.
2) Add 2.5 times the volume of anhydrous ethanol, mix and place at 70 °C for 30 mim.
3) Centrifuge at 12000 g for 5 min and carefully remove the clear.
4) Wash the precipitate with 1 ml of 70% ethanol, dry, then dissolve in 10~20ul of TE buffer or water and store at 70℃.
5. Determine the isotope incorporation ratio in the RNA transcription product and the specific activity of the RNA probe.
1) Dilute the labeled probe with 19ul of nuclease-free water, take 3ul of the diluted sample and dispense it onto 4 DE81 membranes, and allow the membranes to dry at room temperature or under a lamp.
2) Place two membranes directly into separate scintillation vials, add scintillation solution and count. Calculate the average cpm (counts per minute) value for each membrane and determine the cpm value per microliter of the starting reaction using the formula below:
Total cpm/ul for the starting reaction = average cptnX per membrane (20-fold dilution/3ul)
3) Wash off the unbound nucleotides from the remaining two membranes, wash three times with 50~100 ml,0. 5mol/LNa2HPO4 ( pH7.0), drain and put into 70% ethanol, remove and dry at room temperature or under a lamp.
4) Put the membrane into a scintillation vial and add scintillation solution for counting. Calculate the cpm per microliter of RNA bound in the starting reaction:
cpm/ul bound in the starting reaction = average cpm per membrane X (20-fold dilution/3ul)
This formula is also suitable for estimating the specific activity of isotope-labeled RNA probes.
5) Calculate the percent binding from steps 2) and 4).
Percent binding = (cpm bound/total cpm) X 100%.
The binding percentage calculated in this way is usually in the range of 70% to 100%, and low binding of radiolabeled nucleotides (e.g., less than 50%) indicates that RNA synthesis is less efficient.
6) Calculate the specific activity of the probe in terms of cpm/ugRNA synthesized. This is done by first calculating the total bound Cpm
Total bound cpm = (bound cpm/ul)X20ul reaction volume
The next step is to count the total nanomoles of nucleotides in the reaction to determine how many milligrams of RNA were synthesized;
50uCi [ α32 )CTP (400uCi/nmol) equals 0.121 nmol 32P-CTP/reaction. Add 12umol/L unlabeled CTP (0.24nmOl) until CTP reaches 0.36nmol, and if maximal 100% binding is obtained, then the CTP in the RNA transcription product or labeled RNA probe represents ¼ of the nucleotide, and the total amount of nucleotide binding in the probe would be (0.36nmolX4) or 1.44nmol, with an estimated average molecular mass of 330 Da for nucleotides. The average molecular mass of the nucleotide is estimated to be 330 Da, so the amount of RNA synthesized in the sample is 1.44nmolX(330ng/nmol)-475ng. If the binding rate calculated from step 5) is 80%, then the amount synthesized in the actual reaction is 475ngX0.80=380ng.
SA = total binding cpm/ugRNA
In this example, SA is equal to the total bound cpm divided by 0.380ug of RNA.
6. Positive control setup and electrophoretic analysis
Positive controls need to be set up to test whether the reagents. The reagents used and the procedure are standardized. Generally, commercial in vitro transcription kits come with a positive control plasmid template. The reaction system is similar to cyanogen, except that the template is replaced and no isotope-labeled nucleotides are added. Promega's RiboProbe In Vitro Transcription System provides a positive control plasmid that contains three promoters, T7, T3, and SP6, which can be transcribed to yield transcripts of different sizes using different RNA polymerase enzymes.
For RNA electrophoresis, agarose gels or polypropylene-acrylamide gels were prepared using TAE buffer containing no 0.5ug/ml ethidium bromide. Denaturing gels (containing formaldehyde, glyoxal, or 8mol/L urea) can be used to separate denatured RNA, and non-denaturing gels to separate RNA denatured in formaldehyde/formamide RNA sample buffer also give good results.