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Construction of cDNA Expression Library from 20-Week Human Embryonic Brain

Ming Yin

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Abstract

To construct a cDNA expression library from 20-week human embryonic brain, 16.6mg total RNA and 192ug poly(A)+ mRNA have been successively obtained from the 20-week human embryonic brain using single-step method and by chromatography on oligo-(dT) cellulose. With reverse transcriptase (M-MLV), 10μg mRNA was synthesized into 4.4μg blunt cDNA. After the cDNA was ligated to EcoR I adapter and purified by fractionation, 400ng cDNA of about 100bp-8.0kb was collected, of which 50ng cDNA was inserted into λgt11 phage particles. A cDNA library containing 4.6×10 6 recombinants has been constructed. The capacity of this library and the size of cDNA is suitable for futher study.

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What this paper is about

To construct a cDNA expression library from 20-week human embryonic brain, 16.6mg total RNA and 192ug poly(A)+ mRNA have been successively obtained from the 20-week human embryonic brain using single-step method and by chromatography on oligo-(dT) cellulose. With reverse transcriptase (M-MLV), 10μg mRNA was synthesized into 4.4μg blunt cDNA. After the cDNA was ligated to EcoR I adapter and purified by fractionation, 400ng cDNA of about 100bp-8.0kb was collected, of which 50ng cDNA was inserted into λgt11 phage particles. A cDNA library containing 4.6×10 6 recombinants has been constructed. The capacity of this library and the size of cDNA is suitable for futher study.

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Available abstract

To construct a cDNA expression library from 20-week human embryonic brain, 16.6mg total RNA and 192ug poly(A)+ mRNA have been successively obtained from the 20-week human embryonic brain using single-step method and by chromatography on oligo-(dT) cellulose. With reverse transcriptase (M-MLV), 10μg mRNA was synthesized into 4.4μg blunt cDNA. After the cDNA was ligated to EcoR I adapter and purified by fractionation, 400ng cDNA of about 100bp-8.0kb was collected, of which 50ng cDNA was inserted into λgt11 phage particles. A cDNA library containing 4.6×10 6 recombinants has been constructed. The capacity of this library and the size of cDNA is suitable for futher study.

Key concepts: Complementary DNA, cDNA library, Adapter (computing), Reverse transcriptase, Molecular biology, Biology, Messenger RNA, Rapid amplification of cDNA ends

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