User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/24

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(03/24/13)
Current revision (22:54, 24 March 2013) (view source)
(03/24/13)
 
Line 49: Line 49:
* <font color="blue">Pre-warm agar-amp plates with lids closed</font>
* <font color="blue">Pre-warm agar-amp plates with lids closed</font>
-
* <font color="blue">Incubate reactions for '''30 min.'''/ room temp</font>
+
* <font color="blue">Incubate ligations for '''30 min.'''/ room temp</font>
* Add 30 μL DH5α; 5 min/ ice
* Add 30 μL DH5α; 5 min/ ice
* Plate on 100 μg/mL amp; incubate at 37°C
* Plate on 100 μg/mL amp; incubate at 37°C

Current revision

Karmella's BioBrick Cloning Main project page
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03/24/13

  • Assembly do-over: used plain LB agar by mistake last time; use new LB Amp plates


  • Ligations
Ligation Plate results (lig : neg crtl) 03/21/13
1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
4. MV9(X dp)/ 25 ng  
5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase ...
6. MV9(X no dp)/ 25 ng  


  1 2 3 4 9 10
Insert DNA 0.5 1.0 2.0 --- --- ---
Vector DNA 1.0 1.0 1.0 1.0 0.3 0.3
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 ---
dH2O 2.5 2.0 1.0 3.0 3.7 4.7
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL
  • Pre-warm agar-amp plates with lids closed
  • Incubate ligations for 30 min./ room temp
  • Add 30 μL DH5α; 5 min/ ice
  • Plate on 100 μg/mL amp; incubate at 37°C



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