User:Wesley J. Houston/Notebook/MAP/2013/03/18

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(3/18/2013)
Current revision (01:13, 26 March 2013) (view source)
(3/18/2013)
 
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==3/18/2013==
==3/18/2013==
* Retrying ligation of KAH201(S/P)/3403 + KAH182(X/P)/1675
* Retrying ligation of KAH201(S/P)/3403 + KAH182(X/P)/1675
 +
* Concentrations: KAH201 = 48.1 ng/uL, KAH1182 = 6.3 ng/uL
 +
** Concentrations were determined on 2/18/13
-
> Ligations, following corrected version from [[http://openwetware.org/wiki/User:Wesley_J._Houston/Notebook/MAP/2013/02/08|March 4, 2013]], with 50 ng of vector, instead of 25 ng.
+
> Ligations, following corrected version from 3/4/13, with 50 ng of vector instead of 25 ng.
-
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
+
-
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/18/13</font>
+
-
|-
+
-
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 50 ng + KAH182 (X/P)/1675, 50 ng || <font color="blue">TBD</font>
+
-
|-
+
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| 2. KAH201 (S/P)/3403, 50 ng || TBD
+
-
|}
+
-
 
+
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
-
| Insert DNA (KAH182)|| 6.0  || ---  ||
+
| Insert DNA (KAH182)|| 8.0  || ---  ||
|-
|-
| Vector DNA (KAH201)|| 1.0  || 1.0  ||
| Vector DNA (KAH201)|| 1.0  || 1.0  ||
|-
|-
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| 2x lgn buf (Roche) || 5.0  || 5.0  ||
+
| 2x lgn buf (Roche) || 10.0  || 5.0  ||
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
Line 31: Line 25:
| dH<sub>2</sub>O    ||  0  || 3.0  ||
| dH<sub>2</sub>O    ||  0  || 3.0  ||
|-
|-
-
| &nbsp;            || 13 μL || 10 μL ||
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| &nbsp;            || 20 μL || 10 μL ||
|}
|}
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* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
-
 
+
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
 +
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/18/13</font>
 +
|-
 +
| 1. KAH201 (S/P)/<font color="blue">3403</font>, 50 ng + KAH182 (X/P)/1675, 50 ng || <font color="blue">0</font>
 +
|-
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| 2. KAH201 (S/P)/3403, 50 ng || 0
 +
|}
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

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3/18/2013

  • Retrying ligation of KAH201(S/P)/3403 + KAH182(X/P)/1675
  • Concentrations: KAH201 = 48.1 ng/uL, KAH1182 = 6.3 ng/uL
    • Concentrations were determined on 2/18/13

> Ligations, following corrected version from 3/4/13, with 50 ng of vector instead of 25 ng.

  1 2
Insert DNA (KAH182) 8.0 ---
Vector DNA (KAH201) 1.0 1.0
2x lgn buf (Roche) 10.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 0 3.0
  20 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) 03/18/13
1. KAH201 (S/P)/3403, 50 ng + KAH182 (X/P)/1675, 50 ng 0
2. KAH201 (S/P)/3403, 50 ng 0


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