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

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----
'''Assembly'''
'''Assembly'''
* BD006/MV2: insert BD006(E/S)/4182 + vector MV2(E/X)/4529
* Use Roche 2x buffer and NEB T4 ligase
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/20
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/20
* BD006/MV2: BD006(E/S),




'''Ligations'''
'''Ligations'''
# 10 uL rxn, vector + insert
# 10 uL rxn, vector
# 15 uL rxn, vector + insert
# 15 uL rxn, vector


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | 1
| bgcolor=#cfcfcf | 2
| bgcolor=#cfcfcf | 3
| bgcolor=#cfcfcf | 4
|-
|-
| DNA insert || 20.0 μL
| DNA insert || 3.0 || --- || 5.5 || ---
|-
|-
| 10x buffer || 3.0
| DNA vector || 1.5 || 1.5 || 1.5 || 1.5
|-
|-
| enzyme 1 || 1.0
| 2x buffer || 5.0 || 5.0 || 7.5 || 7.5
|-
|-
| enzyme 2 || 1.0
| T4 ligase || 0.5 || 0.5 || 0.5 || 0.5
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 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




Revision as of 17:35, 26 March 2015

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Entry title

  • Plasmid miniprep - 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 ---
  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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