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

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(Autocreate 2012/11/30 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
(11/30/12)
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==mm/dd/yy==
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==11/30/12==
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* Line item 1
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* Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment
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* Line item 2
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-
 
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----
 
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'''Minipreps'''<br>
 
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> Check with E/P digests
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
 
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|-valign="top"
 
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| <u>Reagent</u> || <u>Volume</u>
 
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
 
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| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
 
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|-
 
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| DNA(plasmid) || 2.0 μL
 
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|-
 
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| 10X buffer || 1.5
 
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|-
 
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| EcoRI || 1.0
 
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|-
 
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| PstI || 1.0
 
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|-
 
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| dH<sub>2</sub>O || 9.5
 
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|-
 
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
 
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|}
 
----
----
'''Assemblies'''
'''Assemblies'''
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(X/P)/4529
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
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--> Specific notes<br>
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
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| 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] -->
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| 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 || ---
|-
|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}
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> Measure conc.'s
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> Measure conc.'s of gel purified fragments
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
|-
|-
| <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>
|-
|-
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| 1. Digested part (a/b) || --- || --- || ---
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| 1. KAH3 (X/P) || 0.028 || 1.96 || 28.41
|-
|-
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| 2. Digested part (c/d) || --- || --- || ---
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| 2. MV2 (S/P) || 0.085 || 1.93 || 85.02
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|}
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> Dephosphorylation (Roche)
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
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|-
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| <u>Reagent</u> || <u>Volume</u>
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|-
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| DNA (clean digest) || up to 17 μL (500 ng)
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|-
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| 10x buffer d.p. || 2.0
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|-
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| phosphatase || 1.0
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|-
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| dH<sub>2</sub>O || ---
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-
|-
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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|}
|}
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> Ligations
> Ligations
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
{| 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> mm/dd/yy</font>
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| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
|-
|-
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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| 1. KAH3 (X/P)/751, 8 ng + MV2 (S/P)/4529, 25 ng || <font color="blue">KAH3/MV2 >10:1 (Pick 2)</font>
|-
|-
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| 2. vector(c/d)/ ## ng || &nbsp;
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| 2. MV2 (S/P)/ 25 ng || zero colonies
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
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| Insert DNA        || ### || ---  ||
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| Insert DNA        || 0.3 || ---  ||
|-
|-
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| Vector DNA        || ### || ### ||
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| Vector DNA        || 0.3 || 0.3 ||
|-
|-
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| 2x lgn buf (Roche) || ### || ### ||
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| 2x lgn buf (Roche) || 5.0 || 5.0 ||
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
|-
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| dH<sub>2</sub>O    || ### || ### ||
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| dH<sub>2</sub>O    || 3.4 || 3.7 ||
|-
|-
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| &nbsp;            || # μL || # μL ||
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| &nbsp;            || 10 μL || 10 μL ||
|}
|}
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----
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* Follow quick transformation protocol
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'''Oligo annealing'''
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* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
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# New BB 1
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# New BB 2
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
 
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|-
 
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| 10x annealing buffer || 2.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
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|}
 

Revision as of 17:11, 1 December 2012

Karmella's BioBrick Cloning Main project page
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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/(X/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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