User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/11: 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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==mm/dd/yy==
==01/11/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
* Golden Gate assembly trial 1: mini preps/ digests
* Line item 2
* Golden gate assembly trial 2: transformation




----
----
'''Minipreps'''<br>
'''Golden gate trial 1, Minipreps'''<br>
* Check with E/P digests
* Check with E/P digests


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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u><br>1-4. hPCD-BL01/pSB1A3 = 2686, 2000
| 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:KAH011113_gel1.jpg‎|200px|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) || 3.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 1.5
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| PstI || 1.0
| PstI || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 8.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
 
----
'''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)
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| &nbsp; || 15 μL
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <!-- [[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
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
 
 
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| 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
|}
|}
*  --> 37°C/ ~15 min.


* None of the digests show both expected bands; probably just vector


* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <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;
|}
{| {{table}} 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'''
'''Golden gate assembly trial 2'''
# New BB 1
* Add 10.0 μL assembly reaction to 50.0 μL DH5α-Turbo; ice/ 5 min.; plate on amp agar
# 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 22:21, 26 September 2017

Karmella's BioBrick Cloning Main project page
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01/11/13

  • Golden Gate assembly trial 1: mini preps/ digests
  • Golden gate assembly trial 2: transformation


Golden gate trial 1, Minipreps

  • Check with E/P digests
Reagent Volume Expected:
1-4. hPCD-BL01/pSB1A3 = 2686, 2000 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL
  • --> 37°C/ ~15 min.
  • None of the digests show both expected bands; probably just vector


Golden gate assembly trial 2

  • Add 10.0 μL assembly reaction to 50.0 μL DH5α-Turbo; ice/ 5 min.; plate on amp agar



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