18 cDNA Libraries
285
counts precipitated). The firststrand yield is calculated using the following equations:
% incorporation = incorporated cpm x 100%
total cpm
nmoles dNTP incorporated = dNTP [nmoles/J.ll] x reaction
vol. [J.ll] x% incorp./100
cDNA synthesized [ng] = nmoles DNTP incorporated x 330 ng/nmole
% mRNA converted to cDNA = ng cDNA s_rnthesi~ed
ng mRNA m reactwn
2. Second strand synthesis is performed in two steps:
a) RNAse H is used to partially degrade the RNA components of the
cDNA/RNA hybrids. The remaining RNA fragments serve as primers
for second strand synthesis by DNA polymerase I.
b) E. coli DNA ligase is added to covalently link the newly synthesized
cDNA, and T4 DNA polymerase is used to create blunt ends. Chimerk
clones are not produced by this reaction because E. coli DNA ligase will
not ligate blunt end molecules.
The second strand is synthesized without methylated dNTPs so that the
resultant products are hemi-methylated. This is accomplished by one of
two methods. One approach is to use a minimal concentration of methyldCTP/other dNTPs in the firststrand reaction and a much larger concentration of unmethylated dNTPs for the second reaction. The reaction
kinetics are such that the probability of methylating the Xhol primeradapter during synthesis of the second strand is very low. Another approach is to remove the methyl-dCTP from the reaction mixture using
spin columns or by purifying the cDNA by ethanol precipitation after
completing the firststrand synthesis. Hemi-methylation is important because it protects internal Xhol restriction sites from subsequent restriction. By removing the methyl-dCTP from the second reaction, the Xhol
primer-adapter remains unmethylated and susceptible to cleavage.
Calculations of second strand yield are similar to those described above.
The concentration of the dNTPs, however, will depend on the method
used to synthesize an unmethylated second strand. dNTPs from the initial reaction must be included in the calculation if they are not removed
by precipitation or centrifugation.Aliquots of the first and second strand
reaction products are electrophoresed to determine average fragment
length.
285
counts precipitated). The firststrand yield is calculated using the following equations:
% incorporation = incorporated cpm x 100%
total cpm
nmoles dNTP incorporated = dNTP [nmoles/J.ll] x reaction
vol. [J.ll] x% incorp./100
cDNA synthesized [ng] = nmoles DNTP incorporated x 330 ng/nmole
% mRNA converted to cDNA = ng cDNA s_rnthesi~ed
ng mRNA m reactwn
2. Second strand synthesis is performed in two steps:
a) RNAse H is used to partially degrade the RNA components of the
cDNA/RNA hybrids. The remaining RNA fragments serve as primers
for second strand synthesis by DNA polymerase I.
b) E. coli DNA ligase is added to covalently link the newly synthesized
cDNA, and T4 DNA polymerase is used to create blunt ends. Chimerk
clones are not produced by this reaction because E. coli DNA ligase will
not ligate blunt end molecules.
The second strand is synthesized without methylated dNTPs so that the
resultant products are hemi-methylated. This is accomplished by one of
two methods. One approach is to use a minimal concentration of methyldCTP/other dNTPs in the firststrand reaction and a much larger concentration of unmethylated dNTPs for the second reaction. The reaction
kinetics are such that the probability of methylating the Xhol primeradapter during synthesis of the second strand is very low. Another approach is to remove the methyl-dCTP from the reaction mixture using
spin columns or by purifying the cDNA by ethanol precipitation after
completing the firststrand synthesis. Hemi-methylation is important because it protects internal Xhol restriction sites from subsequent restriction. By removing the methyl-dCTP from the second reaction, the Xhol
primer-adapter remains unmethylated and susceptible to cleavage.
Calculations of second strand yield are similar to those described above.
The concentration of the dNTPs, however, will depend on the method
used to synthesize an unmethylated second strand. dNTPs from the initial reaction must be included in the calculation if they are not removed
by precipitation or centrifugation.Aliquots of the first and second strand
reaction products are electrophoresed to determine average fragment
length.