286
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
lL
d
d ·
· ·
incorporated cpm
~o secon stran mcorporatuon = total cpm
nmoles dNTP incorporated = {(dNTP [nmoles/J.ll] x reaction vol. [J.ll]) -
nmoles incorporated in the first strand
reaction} x o/o
second incorp./100
second strand cDNA synthesized [ng] = nmoles dNTP incorporated x 330
nglnmole
o
·
d bl
d DNA _ second strand cDNA synthesized[ng]
Yo converswn to ou e-stran c
- first strand cDNA synthesized [ng]
3. EcoRI adaptors are Iigated to the blunt-ends by T4 DNA ligase. Excess
linkers are removed by centrifugation through a spin column.
4. The products are prepared for ligation to the vector by cleavage with
Xhol. The Xhol site contributed by the 3' end primer-adapter is the
only unmethylated site in each molecule, and its digestion yields molecules with vector-compatible EcoRI-Xhol cohesive ends. Small end-fragments are removed from the mix by precipitation or spin column centrifugation, and the pool is now ready for ligation. 10 J.lg of mRN A yields
on average 14 J.tg of double-stranded product suitable for cloning. 100 ng
of vector-ready cDNA yields 1 x 10 6 primary recombinants.
Cloning cDNA into Iambda bacteriophage
1. Digest a suitable bacteriophage vector (ZAP Express [Stratagene], Lambda Zap II [Stratagene]) with EcoRI and Xhol. Treat with alkaline phosphatase and remove the stuffer fragments by size fractionation. Resuspend the arms at a concentration of 1 J.tg/J.ll. Pre-prepared vector arms are
also commercially available. Add 100 ng of cDNA containing EcoRI and
Xhol ends (5 J.tg/ml final), 10 J.ll ofvector, 2 J.ll of 10 xligase buffer and
adjust with H20 to a final volume of 19 J.ll. Pipet gently and add 1 J.ll ( 400
U) ofT4 DNA ligase (20 U/J.ll). Pipet again gently and incubate overnight
at 4°C. Inoculate appropriate medium supplemented with MgS04 and
maitose with a suitable E. coli host (eg. XLl-Blue MRF' [Stratagene]).
2. Inoculate 50 ml ofMgS04- and maltose-supplemented broth with 0.5 ml
of the overnight host culture and allow to grow to an OD600 of 0.5- 1.0.
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
lL
d
d ·
· ·
incorporated cpm
~o secon stran mcorporatuon = total cpm
nmoles dNTP incorporated = {(dNTP [nmoles/J.ll] x reaction vol. [J.ll]) -
nmoles incorporated in the first strand
reaction} x o/o
second incorp./100
second strand cDNA synthesized [ng] = nmoles dNTP incorporated x 330
nglnmole
o
·
d bl
d DNA _ second strand cDNA synthesized[ng]
Yo converswn to ou e-stran c
- first strand cDNA synthesized [ng]
3. EcoRI adaptors are Iigated to the blunt-ends by T4 DNA ligase. Excess
linkers are removed by centrifugation through a spin column.
4. The products are prepared for ligation to the vector by cleavage with
Xhol. The Xhol site contributed by the 3' end primer-adapter is the
only unmethylated site in each molecule, and its digestion yields molecules with vector-compatible EcoRI-Xhol cohesive ends. Small end-fragments are removed from the mix by precipitation or spin column centrifugation, and the pool is now ready for ligation. 10 J.lg of mRN A yields
on average 14 J.tg of double-stranded product suitable for cloning. 100 ng
of vector-ready cDNA yields 1 x 10 6 primary recombinants.
Cloning cDNA into Iambda bacteriophage
1. Digest a suitable bacteriophage vector (ZAP Express [Stratagene], Lambda Zap II [Stratagene]) with EcoRI and Xhol. Treat with alkaline phosphatase and remove the stuffer fragments by size fractionation. Resuspend the arms at a concentration of 1 J.tg/J.ll. Pre-prepared vector arms are
also commercially available. Add 100 ng of cDNA containing EcoRI and
Xhol ends (5 J.tg/ml final), 10 J.ll ofvector, 2 J.ll of 10 xligase buffer and
adjust with H20 to a final volume of 19 J.ll. Pipet gently and add 1 J.ll ( 400
U) ofT4 DNA ligase (20 U/J.ll). Pipet again gently and incubate overnight
at 4°C. Inoculate appropriate medium supplemented with MgS04 and
maitose with a suitable E. coli host (eg. XLl-Blue MRF' [Stratagene]).
2. Inoculate 50 ml ofMgS04- and maltose-supplemented broth with 0.5 ml
of the overnight host culture and allow to grow to an OD600 of 0.5- 1.0.