Rice penetrated root cDNA Library

(Deshui Zhang, Henry T. Nguyen lab, TTU, 26 September 2000)

 

Source of RNA:

-          Plant: Rice (Oryza sativa L.) cv  CT9993

-          Type of tissue: Root

-          Growth conditions: greenhouse; TTU.

-          Samples: Penetrated roots (0.5-1.0 cm) were harvested one day after penetration and snap frozen. By Varapong.

-          Total RNA purification: acid guinidium thioanyte phenol chloroform method

-          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 hg of cDNA and 1 mg of vector

-          Packaging: Gigapack III Gold Packaging Extract (Stratagene)

-          Lambda ZAP yield: 3.4 x 107 pfu in 500 ml SM buffer.

-          Non-recombinants (dark blue): not determined.

 

Amplified l Phage Library:

-          Complexity: 6.8 x 106 l ZAP pfu (100 ml of primary library)

-           Amplified the Uni ZAp XR library according to the manual instruction(Stratagene)

-          Lambda ZAP yield: 1.7 x 109 pfu

-           

Mass Excision Phagemid Library:

-          Complexity: 6.8 x 106 l ZAP pfu (100 ml of primary library) added to 900 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 1011 pfu; 100 ml

-          Addition of adsorbed mixture of above ingredients to 40 ml LB medium, 37°C

-          Mass excision performed for ~3 hrs and centrifuged to create a cell pellet and supernatant

-          Supernatant heated at 70°C for 20 min; this is the “Low Amplification” library (L).

-          Cell pellet resuspended in 40 ml fresh LB and grown at 37°C for an additional 16 ~ 20 hr.

-          Supernatant heated at 70°C for 20 min; this is the “High Amplification” library (H).

-          Titered on SOLR (Stratagene) cells:

 

 

 

 

 

 

 

Low Amplification

High Amplification

Phagemid yield (cfu/ml)

6.8 x 105

2.8 x 106

Total phagemid yield (cfu/40ml)

2.72 x 107

1.12 x 108

Amplification (cfu/plaque)

40

1.64 x 102

Percent dark blue colonies

~ 1.5%

 

 

-          Non-recombinants: No clones out of 47 randomly chosen clones contained no insert

 

-          Insert size distribution: