User:Karmella Haynes/Notebook/BioBrick cloning/2012/11/30

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Current revision (22:56, 19 March 2013) (view source)
(11/30/12)
 
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----
----
'''Assemblies'''
'''Assemblies'''
-
# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529
+
# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529
> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
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|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
-
| rowspan="9" | [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
+
| rowspan="9" | [[Image:KAH113012_gel1.jpg‎ |150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || up to 25 μL
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| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
|-
|-
-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
+
| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}
Line 42: Line 42:
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
|-
-
| 1. KAH3 (X/P) || --- || --- || ---
+
| 1. KAH3 (X/P) || 0.028 || 1.96 || 28.41
|-
|-
-
| 2. MV2 (S/P) || --- || --- || ---
+
| 2. MV2 (S/P) || 0.085 || 1.93 || 85.02
|}
|}
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
|-
|-
-
| 1. KAH3 (X/P)/751, ## ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 #:1 (Pick #)</font>
+
| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
|-
|-
-
| 2. MV2 (S/P)/ 25 ng || &nbsp;
+
| 2. MV2 (S/P)/ 25 ng || zero colonies
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
-
| Insert DNA        || ### || ---  ||
+
| Insert DNA        || 0.3 || ---  ||
|-
|-
-
| Vector DNA        || ### || ### ||
+
| Vector DNA        || 0.3 || 0.3 ||
|-
|-
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
Line 68: Line 68:
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
|-
-
| dH<sub>2</sub>O    || ### || ### ||
+
| dH<sub>2</sub>O    || 3.4 || 3.7 ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||
|}
|}
 +
 +
* Follow quick transformation protocol
 +
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

11/30/12

  • Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment



Assemblies

  1. KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529

> Digests (Fermentas FD)


Reagent Volume   Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH3 (X/P) 0.028 1.96 28.41
2. MV2 (S/P) 0.085 1.93 85.02


> Ligations

Ligation Plate results (lig : neg crtl) 11/31/12
1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng KAH3/MV2 >10:1 (Pick 2)
2. MV2 (S/P)/ 25 ng zero colonies
  1 2
Insert DNA 0.3 ---
Vector DNA 0.3 0.3
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 3.4 3.7
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar



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