Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/18"

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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>}}
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|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. ##### -->
 
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==mm/dd/yy==
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==04/18/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
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* MV9 cloning - sequencing order
* Line item 2
 
  
  
 
----
 
----
'''Minipreps'''<br>
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'''MV9 sequencing'''<br>
* Check with E/P digests
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* DNASU
 
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** DNA: 200 ng/ 10 μL
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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** Primers: 16 pmol (16 μM) / 10 μL
|- valign="top"
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Volume
 
| 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] -->
 
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| DNA(plasmid) || 2.0 μL
 
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| 10X buffer || 1.5
 
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| EcoRI || 1.0
 
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| PstI || 1.0
 
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| dH<sub>2</sub>O || 9.5
 
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| &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"
 
| bgcolor=#cfcfcf | Reagent
 
| 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
 
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| 10x buffer || 3.0
 
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| enzyme 1 || 1.0
 
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| enzyme 2 || 1.0
 
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| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
|}
 
  
  
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| Sample || OD260 || 260/280 || ng/μL
 
| Sample || OD260 || 260/280 || ng/μL
 
|-
 
|-
| 1. Digested part (a/b) || --- || --- || ---
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| 1. 1-3 || 0.054 || 1.825 || 54.37
 
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| 2. Digested part (c/d) || --- || --- || ---
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| 2. 3-5 || 0.047 || 1.73 || 47.14
|}
 
 
 
 
 
* 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
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| 3. 3-6 || 0.131 || 1.87 || 130.79
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
 
 
|}
 
|}
  
  
* Ligations
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* Sequencing results for order# 7177
{| {{table}} cellspacing="3" <!-- Ligations table -->
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** MV9 1-3/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
|- bgcolor=#cfcfcf
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** MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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** MV9 3-6/ DD122B (fwd): correct sequence but backwards; reads through DD123B (rev primer) on other side
|-
 
| 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        || ###  || ---  ||
 
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| Vector DNA        || ###  || ###  ||
 
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| 2x lgn buf (Roche) || ###  || ###  ||
 
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
|-
 
| dH<sub>2</sub>O    || ###  || ###  ||
 
|-
 
| &nbsp;            || # μL || # μL ||
 
|}
 
  
----
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* Keep MV9 1-3 in vector stocks. See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/04/12 colony PCR from 04/12/13] for gel confirmation.
'''Oligo annealing'''
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* Keep MV9 3-6 in case it might be useful
# 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 21:39, 26 September 2017

Owwnotebook icon.pngKarmella's BioBrick Cloning Report.pngMain project page
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04/18/13

  • MV9 cloning - sequencing order



MV9 sequencing

  • DNASU
    • DNA: 200 ng/ 10 μL
    • Primers: 16 pmol (16 μM) / 10 μL


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 1-3 0.054 1.825 54.37
2. 3-5 0.047 1.73 47.14
3. 3-6 0.131 1.87 130.79


  • Sequencing results for order# 7177
    • MV9 1-3/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
    • MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
    • MV9 3-6/ DD122B (fwd): correct sequence but backwards; reads through DD123B (rev primer) on other side


  • Keep MV9 1-3 in vector stocks. See colony PCR from 04/12/13 for gel confirmation.
  • Keep MV9 3-6 in case it might be useful