User:Wesley J. Houston/Notebook/MAP/2013/03/13: Difference between revisions

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==3/13/13==
==3/13/13==
* Attempt ligation again with original plasmids.
* Attempted ligation following the same protocol outlined on 3/4/13.
* Grow liquid cultures of KAH182 in case this ligation attempt fails.
* Grew four liquid cultures of KAH182, following the same procedure (pick a colony and place into LB broth), in case this ligation attempt fails.


> Ligations
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA (KAH182)        || 3.0  || ---  ||
|-
| Vector DNA (KAH201)        || 0.5  || 0.5  ||
|-
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    ||  0.5 ||  3.5 ||
|-
| &nbsp;            || 10 μL || 10 μL ||
|}
* Follow quick transformation protocol
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
> Ligations (corrected version, 3/04/13)
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/13/13</font>
|-
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font>
|-
| 2. KAH201 (S/P)/3403, 25 ng || 0
|}


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Revision as of 22:12, 25 March 2013

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3/13/13

  • Attempted ligation following the same protocol outlined on 3/4/13.
  • Grew four liquid cultures of KAH182, following the same procedure (pick a colony and place into LB broth), in case this ligation attempt fails.

> Ligations

  1 2
Insert DNA (KAH182) 3.0 ---
Vector DNA (KAH201) 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 0.5 3.5
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar

> Ligations (corrected version, 3/04/13)

Ligation Plate results (lig : neg crtl) 03/13/13
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 0
2. KAH201 (S/P)/3403, 25 ng 0


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