Primer extension

목차

1. Introduction
2. Materials
3. Method
References

본문내용

primer extension analysis is utilized to quantitate mRNA levels, and to detect low abundance mRNA species. In addition, primer extension analysis can also be utilized to map the 5'-end of transcripts to determine the exact start site(s) for transcription.

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2. Add 500ul of TE (pH 7.6) to stop kinase reaction. Add 25 ug of carrier RNA
3. Phenol/Chloroform extraction
: add equilibrated phenol (pH 8.0) 400 ul + chloroform 400 ul
-> vigorous vortexing for 20 sec, centrifuge for 2 min
-> transfer aqueous layer to a fresh sterile microfuge tube
4. Chloroform extraction
: add chloroform 800 ul
-> vigorous vortexing for 20 sec, centrifuge for 2 min
-> transfer aqueous layer to a fresh sterile microfuge tube

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19. Centrifuge at 4℃ for 10 min, remove sup.
-> carefully rinse the pellets with 400 ul of 70% EtOH,
-> centrifuge at 4℃ for 5 min, carefully remove sup. with pipette
-> dry the tubes in the air
20. Resuspend the pellets in 10 ul of formamide loading buffer with pipetting

참고 자료

Sambrook and Russel (2003), Molecular Cloning (3rd Ed.), vol 1 7.75-7.81
Krug, M.S., and Berger, S.L. (1987) Meth. Enzymol. 152, 316
Freeman, W.M., Vrana, S.L., and Vrana, K.E. (1996) BioTechniques 20, 782.