Molecular Techniques and Methods

Plasmid Transformation by Triparental Conjugation into Agrobacterium


Copy Right © 2001/ Institute of Molecular Development LLC



INTRODUCTION




MATERIALS AND SOLUTIONS

YEM medium
Yeast extract ---------------------- 0.4 g
20 x Salts ------------------------- 50 ml
Mannitol --------------------------- 10 g
Agar ------------------------------- 10 g


20 x Salts for YEM
K2HPO4 ------------------------- 1 g
MgSO4.7H2O --------------------- 0.4 g
NaCl ---------------------- 0.2 g
H2O to make ----------------------- 100 ml

  • Adjust pH to 6.9.
  • Autoclave.


    Minimal T Medium
    Agar ----------------------------------- 5 g
    H2O ------------------------- 435 ml
  • Autoclave.

    Add the following sterile solutions.
    20 x Salts ------------------------- 25 ml
    20 x K-P buffer -------------------- 25 ml
    20% Sucrose solution --------------- 12.5 ml
    Kanamycin -------------------------- 500 ug


    20 x Salts for Minimal T
    NH4Cl --------------------- 2 g
    MgSO4.7H2O --------------------- 0.4 g
    MnCl2 --------------------- 0.004 g
    CaCl2 --------------------- 0.02 g
    FeSO4.7H2O ----------------- 0.01 g
    H2O to make ---------------------- 100 ml
  • Autoclave.


    20 x K-P Buffer
    K2HPO4 ------------------ 21 g
    KH2PO4 ------------------ 9 g
    H2O to make -------------------- 100 ml
  • Autoclave.




  • PROCEDURES

    1. Day 1: Start Agrobacteriumin 5 ml selective media (ie., YEM) and grow 2 days at 28-30o.

    2. Day 2: Start following E.coli strains in 1 ml selective media.
    E.coli (pRK2013) in LB + kanamycin
    E.coli (pBI) in LB + kanamycin

    3. Days 3: Spin down all three cultures at 1,000 g for 5 min.

    4. Resuspend cells in 1 ml LB.
    This removes antibiotics and concentrate the Agrobacterium.

    5. Take 0.2 ml of each resuspended culture and mix together in sterile microfuge tubes.

    6. Vortex briefly.
    Spin down cells at 1,000g for 5 min.

    7. Resuspend cells in 100 ul LB.

    8. Plate on LB plates and incubate at 28-30o over night.

    9. Streak a loopful of bacteria from each plate onto
    Minimal T with 0.5% sucrose and kanamycin.

    10. Incubate at 28-30o for 3-5 days.



    NOTES




    KIT INFORMATION


    REFERENCES


    Please send your comment on this protocol to "editor@MolecularInfo.com".

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