User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2013/01/09 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(fix raw html notebook nav)
 
(7 intermediate revisions by one other user not shown)
Line 2: Line 2:
|-
|-
|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}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### 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==
==01/09/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Golden Gate assembly: hPCD + BL01 + pSB1A3
* Line item 2
* Violacein: plates from Tabor; incubate o/n




----
----
'''Minipreps'''<br>
'''Golden Gate Assembly'''<br>
* Check with E/P digests
* Use purified, measured PCR products from 1/05/13
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
* Dilutions
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | length (bp)
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| bgcolor=#cfcfcf | ng/μL
| 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] -->
| bgcolor=#cfcfcf | Vol.
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
| DNA(plasmid) || 2.0 μL
| gg2 pSB1A3 || 2000 || 67.7 || 7.7 || 12.3
|-
|-
| 10X buffer || 1.5
| gg3 hPCD || 186 || 22.5 || 2.1 || 17.8
|-
|-
| EcoRI || 1.0
| gg4 BL01 || 2520 || 55.9 || 11.7 || 8.3
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &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


* Golden Gate Reactions
# hPCD + BL01 + pSB1A3
# pSB1A3


* Digests (Fermentas FD)
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| Reagent || 1 || 2
| 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
|}
 
 
* 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>
| gg2 pSB1A3 || 1.0 || 1.0
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| gg3 hPCD || 1.0 || ---  
|}
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
|-
| Insert DNA        || ###  || --- ||
| gg4 BL01 || 1.0 || ---  
|-
|-
| Vector DNA        || ###  || ###  ||
| 10x ligase buffer || 1.0 || 1.0
|-
|-
| 2x lgn buf (Roche) || ###  || ###  ||
| NEB T4 lgase || 0.25 || 0.25
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| BsmBI || 0.5 || 0.5
|-
|-
| dH<sub>2</sub>O   || ###  || ###  ||
| dH<sub>2</sub>O || 5.25 || 7.25
|-
|-
| &nbsp;             || # μL || # μL ||
| &nbsp; || 10.0 || 10.0
|}
|}


----
Thermal cycler
'''Oligo annealing'''
* [45°C, 2 min.; 16°C, 5 min.] x25
# New BB 1
* 60°C, 20 min.
# New BB 2
* 80°C, 20 min.
 
{| 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 22:20, 26 September 2017

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

01/09/13

  • Golden Gate assembly: hPCD + BL01 + pSB1A3
  • Violacein: plates from Tabor; incubate o/n


Golden Gate Assembly

  • Use purified, measured PCR products from 1/05/13
  • Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
  • Dilutions
DNA length (bp) ng/μL Vol. + dH2O
gg2 pSB1A3 2000 67.7 7.7 12.3
gg3 hPCD 186 22.5 2.1 17.8
gg4 BL01 2520 55.9 11.7 8.3


  • Golden Gate Reactions
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/09&oldid=1011738"