The RNA template is used to obtain DNA by reverse transcription, which is then used to replicate and infect cells.

Operation method

RNA reverse transcription

Materials and Instruments

poly(A)+RNA reverse transcriptase Mouse-derived reverse transcriptase or avian-derived reverse transcriptase
oligo(dT)12-18 lmol LTris-Cl 1 mol LTris-Cl lmol LKC1 25 mmol LMgCl2 dNTP mixture 0.lmol LDTT RNasin

Move

I Materials and equipment

1) poly(A ^)+RNA, lmg/ml

Z)oligo(dT _)12-18,lmg/ml

3)lmol/LTris-Cl (pH 7.6).

4)1mol/LTris-Cl(pH8.3)

5)lmol/LKC1

6)25 _mmol/LMgCl2

7)dNTP mixture, 5 mmol/L each

8)0.lmol/LDTT

9)RNasin.

10) Reverse transcriptase, mouse-derived reverse transcriptase (Pharmacia, 200 U/ul) or avian-derived reverse transcriptase (20 U/ul)


II. Methods of operation

(a) Synthesize cDNA using murine reverse transcriptase.

1) Mix the following in a sterilized microcentrifuge tube and place on ice.

Poly(A ^)+4RNA, lmg/ml 10ul

oligo(dT _)12-18,lmg/ml 10ul

lmol/LTris-Cl (pH 7.6) 2.5ul

lmol/LKC1 3.5ul

250 _mmol/LMgCl2 2ul

dNTP mixture, 5 mmol/L each 10ul

0.lmol/LDTT 2ul

RNasin 25U

Mouse-derived reverse transcriptase 200U

Add water to a total volume of 50ul

2) React at 37°C for lh

3) Purify and recover. Store at 20°C.

(ii) Synthesis of cDNA using avian reverse transcriptase

1) Mix the following in a pyrotechnic microcentrifuge tube and place the well on ice.

poly(A ^)+RNA, lmg/ml 10ul

oligo(dT)12-18,lmg/ml 10u1

lmol/LTris-Cl (pH 8.3) 2.5uI

lmol/LKC1 3.5ul

250 mmol/LMgCI2 2ul

dNTP mixture, 3 mmol/L each 10ul

0.lmol/LDTT 2ul

RNasin 25U

Avian-derived invertase 40U

Add water to a total volume of 50ul

2) React at 42℃ for lh

3) Purify the cell and store at 20°C.

Caveat

1) A small pre-experiment can be performed first to determine the amount of reverse transcriptase.2) Add to the reaction [α32P]dCTP in the reaction, the quality and yield of reverse transcription can be detected by radioautography.3) For transcription of mRNAs with secondary structures that are detrimental to the transcription reaction, the use of avian reverse transcriptase with an optimal temperature of 42°C may yield better results.4) In addition to the use of oligo(dt)12-18In addition to the use of oligo(dt) 12-18, random oligonucleotides of length 6 can also be used in the reverse transcription.5) With the development and gradual popularization of gene chip technology, the method of obtaining fluorescently labeled cDNA probes from mRNA reverse carving has been developed. The difference with the commonly used isotope labeling of RNA probes is the addition of the fluorescent dyes Cy3 or Cy5 instead of the dNTP labeling of the model.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/