Synthesis of long cDNA from viral RNA template

J. A. Lenstra; R. J. De Groot; L. Jacobs; J. G. Kusters; H. G M Niesters; B. A M Van Der Zeijst

doi:10.1016/0735-0651(88)90017-9

Synthesis of long cDNA from viral RNA template

J. A. Lenstra^*
, R. J. De Groot
, L. Jacobs
, J. G. Kusters
, H. G M Niesters
, B. A M Van Der Zeijst

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

Original language	English
Pages (from-to)	57-61
Number of pages	5
Journal	Gene Analysis Techniques
Volume	5
Issue number	3
DOIs	https://doi.org/10.1016/0735-0651(88)90017-9
Publication status	Published - 1 Jan 1988

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title = "Synthesis of long cDNA from viral RNA template",

abstract = "Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.",

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AU - Lenstra, J. A.

AU - De Groot, R. J.

AU - Jacobs, L.

AU - Kusters, J. G.

AU - Niesters, H. G M

AU - Van Der Zeijst, B. A M

PY - 1988/1/1

Y1 - 1988/1/1

N2 - Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

AB - Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

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VL - 5

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EP - 61

JO - Gene Analysis Techniques

JF - Gene Analysis Techniques

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ER -

Synthesis of long cDNA from viral RNA template

Abstract

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