Morex Barley
Pre-Anthesis Spike cDNA Library (D-W Choi, TJ Close lab, UC Riverside, 28
October 1999); HVcDNA0008; HVSMEg
Source of
RNA:
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Plant: barley
(Hordeum vulgare L. cv. Morex)
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Type of
tissue: spike
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Growth
conditions: greenhouse; UC Riverside Fall 98 to Winter 99
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Samples: whole
spike with awns trimmed; white, green and yellow anther
stages
-
Total RNA
purification: hot phenol method.
Nucleic Acid Res (1989) 17:2362
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RNA pooling:
equal amounts of total RNA were mixed from all three
samples
-
PolyA
purification method: PolyATtrack mRNA Isolation System IV
(Promega)
-
cDNA synthesis
method: cDNA Synthesis Kit (Stratagene)
Name of
vector: Uni-ZAP XR
-
Uni-ZAP XR
vector allows in vivo excision of the
pBluescript phagemid
Description of
Inserts:
-
cDNAs were
directionally cloned with EcoRI on
the 5’ and XhoI on the 3’
end
-
cDNAs larger
than 0.5 kb were selected by size fractionation via gel
filtration
Primary
l Phage
Library:
-
cDNA ligation:
100 ng of cDNA and 1 mg of
vector
-
Packaging:
Gigapack III Gold Packaging Extract (Stratagene)
-
Lambda ZAP
yield: 1.23 x 107 pfu in 500 ml SM
buffer
-
Non-recombinants
(dark blue): not determined
Mass Excision
Phagemid Library:
-
Complexity: ~1
x 106 l ZAP pfu; 42
µl of primary library added to 958 ml SM
Buffer
-
Host
cells: XL1-Blue-MRF’ (Stratagene),
~ 5 x 107 log-phase cells in 1 ml 10 mM
MgSO4
-
Helper phage:
ExAssist (Stratagene), ~1 x 109 pfu; 100 ml
-
Addition of
adsorbed mixture of above ingredients to 40 ml LB medium, 37°C
-
Mass excision
performed for 3 hrs, stopped by heating to 70°C
-
Phagemid
yield: 3.01 x 106 cfu/ml
on SOLR (Stratagene) cells
-
Amplification:
[(40 ml) x (3.01 x 106 cfu/ml)]/(1 x 106 l ZAP pfu)
= 120
-
Percent dark
blue colonies: ~ 2%
-
Non-recombinants:
1 of 36 randomly chosen clones contained no insert
-
Insert size
distribution:
