plant surface microbiology.pdf

29 Functional Genomic Approaches for Studies of Mycorrhizal Symbiosis 575
ensure that you obtain the best possible library from your cDNA, set up three
parallel ligations using three different ratios of cDNA to vector, as shown below.
1. Label three 0.5-ml tubes and add the indicated reagents. Mix the reagents
gently; avoid producing air bubbles. Spin tubes briefly to bring contents to
the bottom of the tube.
Ligations using three different ratios of cDNA to phage vector
Component 1st ligation 2nd ligation 3rd ligation
cDNA 0.5 1.0 1.5
Vector (500 ng/ml) 1.0 1.0 1.0
10¥ Ligation buffer* 0.5 0.5 0.5
ATP (10 mM) 0.5 0.5 0.5
T4 DNA Ligase 0.5 0.5 0.5
Deionized H 2O 2.0 1.5 1.0
Total volume (ml) 5.0 5.0 5.0
*x10 ligation buffer: 300 mM Tris-HCl, pH 7.8, 100 mM MgCl 2, 100 mM
DTT
2. Incubate tubes at 16 °C overnight.
3.1.6 Packaging of Ligated cDNA and Preparation of cDNA Library
Perform a separate, l-phage packaging reaction for each of the ligations as
per manufacturer’s instructions.We used Gigapack packaging extracts (Stratagene,
CA, USA) and also MaxPlaq packaging extracts (Epicenter, WI, USA)
with very good success.
1. Thaw three packaging extracts (50 ml per extract) on ice.
2. Immediately after the extracts have thawed, add 5 ml of each ligation mixture
to one tube, mix gently.
3. Incubate at 22 °C for 4 h and add phage buffer (20 mM Tris-HCl, pH 7.4;
100 mM NaCl; 10 mM MgSO 4) to 250 ml and 10 ml of chloroform. Gently mix
well and allow the chloroform to settle down. This packaged mix can be
stored at 4 °C up to 4 weeks.
4. Titer each of the resulting libraries. From the three ligations combined, you
should obtain 1–2x10 6 independent clones.
Note:Ifyou obtained