User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/01: Difference between revisions

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(Autocreate 2012/12/01 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==12/01/12==
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* Line item 1
* Cultures/ minipreps: KAH3/MV2 colonies (2)
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----
----
'''Minipreps'''<br>
'''Minipreps'''<br>
> Check with E/P digests
* Check with PstI digest
* Compare to empty vector MV2


{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
| <u>Reagent</u> || <u>Volume</u>  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1. MV2 = 4529<br>2. KAH3/MV2-1 = 5280<br>3. KAH3/MV2-2 = 5280<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] -->
| 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
| DNA(plasmid) || 2.0 μL
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|}
|}


----
'''Assemblies'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
> Digests (Fermentas FD)<br>
--> Specific notes<br>
{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
|-valign="top"
| <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] -->
|-
| DNA (plasmid) || up to 25 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
> Measure conc.'s
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
|-
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 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
|}
> Ligations
{| 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>
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
| 2. vector(c/d)/ ## ng || &nbsp;
|}
{| class="wikitable" border="0" cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}
----
'''Oligo annealing'''
# 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
|}




Revision as of 14:19, 1 December 2012

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12/01/12

  • Cultures/ minipreps: KAH3/MV2 colonies (2)


Minipreps

  • Check with PstI digest
  • Compare to empty vector MV2
Reagent Volume Expected:
1. MV2 = 4529
2. KAH3/MV2-1 = 5280
3. KAH3/MV2-2 = 5280
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.



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