User:Karmella Haynes/Notebook/BioBrick cloning/2012/11/30: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==mm/dd/yy==
==11/30/12==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Assembly: CMVTetO2:K (KAH3)/ MV; expression vector for Vi's mammalian cell expression experiment
* Line item 2


----
'''Minipreps'''<br>
> Check with E/P digests
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}


----
----
'''Assemblies'''
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# KAH3/MV2: (1) KAH3/(X/P)/751 + (2) MV2/(S/P)/4529
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


> Digests (Fermentas FD)<br>
> Digests (Fermentas FD)<br>
--> Specific notes<br>
 


{| 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;
| 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.
|}
|}




> Measure conc.'s
> 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>
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. KAH3 (X/P) || 0.028 || 1.96 || 28.41
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
 
 
> Dephosphorylation (Roche)
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
|-
| <u>Reagent</u> || <u>Volume</u>
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| 2. MV2 (S/P) || 0.085 || 1.93 || 85.02
|}
|}


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> Ligations
> Ligations
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> 11/31/12</font>
|-
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #: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. vector(c/d)/ ## 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) || ### || ### ||
| 2x lgn buf (Roche) || 5.0 || 5.0 ||
|-
|-
| 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;            || # μL || # μL ||
| &nbsp;            || 10 μL || 10 μL ||
|}
|}


----
* Follow quick transformation protocol
'''Oligo annealing'''
* Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
# New BB 1
# New BB 2


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Latest revision as of 22:18, 26 September 2017

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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