Name of vector: Uni-zap xr vector allows in vivo excision of the pBluescript phagemid Description of Inserts




Yüklə 12.4 Kb.
tarix22.04.2016
ölçüsü12.4 Kb.

Aegilops speltoides anther cDNA Library (E Akhunov, P Kianian, K Simons, C Otto, TJ Close, D-W Choi, D Zhang, A Chin, RD Fenton; TJ Close lab, UC Riverside, 4 Aug 2000); AS040E1X; AScDNA0001 Lot #1.



Source of RNA:

  • Plant: diploid aegilops (Aegilops speltoides). F2 plants from a cross of 2-12-4-8-1-1-1-(1) female by PI 36909-12-811-(1) male.

  • Type of tissue: anther

  • Growth conditions: Growth chamber, normal conditions, E. Akhunov, UCD.

  • Samples: premeiotic anthers.

  • Total RNA purification: hot phenol method. Nucleic Acid Res (1989) 17:2362.

  • RNA pooling: one sample

  • PolyA purification method: PolyATtrack mRNA Isolation System III (Promega)

  • cDNA synthesis method: cDNA Synthesis Kit (Stratagene)


Name of vector: Uni-ZAP XR


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  Phage Library:

  • cDNA ligation: 100 ng of cDNA and 1 g of vector

  • Packaging: Gigapack III Gold Packaging Extract (Stratagene)

  • Lambda ZAP yield: 1.8 x 107 pfu in 500 l SM buffer.

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


Mass Excision Phagemid Library:

  • Complexity: 1 x 106  ZAP pfu (28 l of primary library) added to 972 l 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 l

  • 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)

1.17 x 107

7.55 x 108

Total phagemid yield (cfu/40ml)

4.68 x 108

3.02 x 1010

Amplification (cfu/plaque)

4.68 x 102

3.02 x 104

Percent dark blue colonies

~ 2.5

Not determined


Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©azrefs.org 2016
rəhbərliyinə müraciət

    Ana səhifə