# Butte 86 Wheat Developing Grains cDNA Library (A Chin & RD Fenton; TJ Close lab, UC Riverside, May 2001); TAcDNA0025 Lot #1; TA059E1X

**Source of RNA:**

-   Plant: hexaploid wheat (*Triticum aestivum* L. cv Butte 86)

-   Type of tissue: whole grains

-   Growth conditions: plants were grown under six different
    environmental regimes in greenhouses by Susan B. Altenbach and K.
    Cronin, USDA-ARS, Albany, CA.

> Environment 1) 24°C/17°C day/night, well-watered, with post-anthesis
> fertilizer
>
> Environment 2) 24°C/17°C day/night, well-watered, without
> post-anthesis fertilizer
>
> Environment 3) 37°C/17°C day/night, well-watered, with post-anthesis
> fertilizer
>
> Environment 4) 37°C/17°C day/night, well-watered, without
> post-anthesis fertilizer
>
> Environment 5) 37°C/17°C day/night plus drought, with post-anthesis
> fertilizer
>
> Environment 6) 37°C/17°C day/night plus drought, without post-anthesis
> fertilizer

-   Samples: developing wheat grains from the following were excised and
    frozen in liquid nitrogen

> Environment 1 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44
> DPA
>
> Environment 2 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 32, 36, 40, 44
> DPA
>
> Environment 3 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 32, 34 DPA
>
> Environment 4 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 32, 34 DPA
>
> Environment 5 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 30 DPA
>
> Environment 6 at 3, 5, 7, 8, 10, 12, 16, 20, 24, 28, 30 DPA

-   Total RNA purification: TAG 97: 413-421 (1998); K. Cronin in S.
    Altenbach lab.

-   RNA pooling: 10 μg RNA from early time points (3-10 DPA) and 20 μg
    RNA from middle and late time points (12-44 DPA).

-   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 *Eco*RI on the 5' and *Xho*I on
    the 3' end

-   cDNAs larger than 0.5 kb were selected by size fractionation via gel
    filtration

**Primary λ Phage Library:**

-   cDNA ligation: \~50 ng (5/21) and \~ 50 ng (5/23/01) of cDNA and 1
    μg of vector each time

-   Packaging: Gigapack III Gold Packaging Extract (Stratagene); twice.

-   Lambda ZAP yield: 8.4 x 10^5^ pfu (5/23/01) and 4.0 x 10^6^ pfu
    (5/25/01) each in 500 μl SM buffer for a combined total of 4.8 x
    10^6^ pfu in 1 ml.

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

**Mass Excision Phagemid Library:**

-   Complexity: 1 x 10^6^ λ ZAP pfu (209 μl of primary library added to
    791 μl SM Buffer)

-   Host cells: XL1-Blue-MRF\' (Stratagene), \~ 5 x 10^7^ log-phase
    cells in 1 ml 10 mM MgSO~4~

-   Helper phage: ExAssist (Stratagene), \~1 x 10^9^ pfu; 40 μl

-   Addition of adsorbed mixture of above ingredients to 39 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).

-   Titered on TJC121 cells (5/31/2001 by Edmundo Rodriguez):

+----------------------------------------+-----------------------------+
|                                        | Low Amplification           |
|                                        |                             |
|                                        | (Lot \# 1.1.L)              |
+----------------------------------------+-----------------------------+
| Phagemid yield (cfu/ml)                | 2.07 x 10^7^                |
+----------------------------------------+-----------------------------+
| Total phagemid yield (cfu/40ml)        | 8.3 x 10^8^                 |
+----------------------------------------+-----------------------------+
| Amplification (cfu/plaque)             | 828                         |
+----------------------------------------+-----------------------------+
| Percent dark blue colonies             | \~ 4%                       |
+----------------------------------------+-----------------------------+