Haynes Lab:Notebook/Jan/2015/03/26: Difference between revisions

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'''Assembly'''
'''Assembly'''
*'''[[User:Karmella Haynes|---Karmella]] 21:16, 26 March 2015 (EDT)''':
* BD006/MV2: insert BD006(E/S)/4182 + vector MV2(E/X)/4529
* BD006/MV2: insert BD006(E/S)/4182 + vector MV2(E/X)/4529
* Use Roche 2x buffer and NEB T4 ligase
* Use Roche 2x buffer and NEB T4 ligase
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'''Ligations'''
* Ligations
# 10 uL rxn, vector + insert
# 10 uL rxn, vector + insert
# 10 uL rxn, vector
# 10 uL rxn, vector

Revision as of 18:16, 26 March 2015

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03/26/15

  • Plasmid miniprep - (2x) 5 mL liquid culture for DPRE (D11.12)
  • Assembly - BD006 into MV2


Assembly


  • Ligations
  1. 10 uL rxn, vector + insert
  2. 10 uL rxn, vector
  3. 15 uL rxn, vector + insert
  4. 15 uL rxn, vector
Reagent 1 2 3 4
DNA insert 3.0 --- 5.5 ---
DNA vector 1.5 1.5 1.5 1.5
2x buffer 5.0 5.0 7.5 7.5
T4 ligase 0.5 0.5 0.5 0.5
dH2O --- 3.0 --- 5.5
  10 μL 10 μL 15 μL 15 μL

--> RT/ ~30 min.

  • Add 50 μL DH5-α turbo
  • 5 min./ ice
  • Plate on 100 ug/mL amp agar. Grow at 37 °C o/n




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