User:Wesley J. Houston/Notebook/MAP/2013/03/08

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(3/8/13)
Current revision (01:10, 26 March 2013) (view source)
(3/8/13)
 
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* All attempts proved unsuccessful.
* All attempts proved unsuccessful.
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
+
> Ligations
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|-valign="top"
+
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
-
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| &nbsp;            || 1    || 2    ||
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| rowspan="9" |  
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|-
|-
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| DNA (plasmid) || 20 μL
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| Insert DNA (KAH182)       || 3.0  || ---  ||
|-
|-
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| 10x buffer || 3.0
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| Vector DNA (KAH201)        || 0.5  || 0.5  ||
|-
|-
-
| Enzyme 1 (Pst1) || 1.0
+
| 2x lgn buf (Roche) || 5.0 || 5.0  ||
|-
|-
-
| Enzyme 2 (Spe1 or Xba1) || 1.0
+
| T4 ligase (NEB)   || 1.0 || 1.0  ||
|-
|-
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| dH<sub>2</sub>O || 5.0 μL
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| dH<sub>2</sub>O   || 0.5 ||  3.5 ||
|-
|-
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| &nbsp; || 30 μL --> 37°C/ ~15 min.
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| &nbsp;             || 10 μL || 10 μL ||
|}
|}
 +
* Follow quick transformation protocol
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* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
 +
 +
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/08/13</font>
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|-
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| 1. KAH201 (S/P)/<font color="blue">3403</font>, 25 ng + KAH182 (X/P)/1675, 26 ng || <font color="blue">0</font>
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|-
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| 2. KAH201 (S/P)/3403, 25 ng || 0
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|}
 +
 +
* No colonies were present in the ligation plate.
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3/8/13

  • Ligation was repeated.
  • Attempted to grow more liquid cultures of ligated plasmids.
  • Attempted to confirm ligation by streaking the colonies on an agar plate.
  • All attempts proved unsuccessful.

> 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
Ligation Plate results (lig : neg crtl) 03/08/13
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 0
2. KAH201 (S/P)/3403, 25 ng 0
  • No colonies were present in the ligation plate.


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