User:Karmella Haynes/Notebook/BioBrick cloning/2013/04/18

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(Autocreate 2013/04/18 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (15:50, 3 September 2013) (view source)
(04/18/13)
 
(7 intermediate revisions not shown.)
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==mm/dd/yy==
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==04/18/13==
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* Line item 1
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* MV9 cloning - sequencing order
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* Line item 2
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----
----
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'''Minipreps'''<br>
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'''MV9 sequencing'''<br>
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* Check with E/P digests
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* DNASU
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** DNA: 200 ng/ 10 μL
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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** Primers: 16 pmol (16 μM) / 10 μL
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| 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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|-
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| DNA(plasmid) || 2.0 μL
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|-
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| 10X buffer || 1.5
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|-
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| EcoRI || 1.0
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|-
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| PstI || 1.0
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|-
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| dH<sub>2</sub>O || 9.5
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|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
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|}
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----
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'''Assemblies'''
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Digests (Fermentas FD)
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** Specific notes
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| 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] -->
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|-
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| DNA (plasmid) || up to 25 μL
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|-
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| 10x buffer || 3.0
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|-
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| enzyme 1 || 1.0
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|-
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| enzyme 2 || 1.0
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|-
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| dH<sub>2</sub>O || ---
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|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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|}
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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
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|-
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| 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
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|}
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* Dephosphorylation (Roche)
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{| {{table}} cellspacing="3" <!-- Dephos table -->
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|-
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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|-
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| DNA (clean digest) || up to 17 μL (500 ng)
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|-
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| 10x buffer d.p. || 2.0
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| phosphatase || 1.0
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|-
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| dH<sub>2</sub>O || ---
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|-
|-
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| 3. 3-6 || 0.131 || 1.87 || 130.79
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|}
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* Ligations
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* Sequencing results for order# 7177
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{| {{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
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|- bgcolor=#cfcfcf
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** MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side
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| 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
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|-
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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|-
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| 2. vector(c/d)/ ## ng || &nbsp;
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|}
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
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| &nbsp;            || 1    || 2    ||
 
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|-
 
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| Insert DNA        || ###  || ---  ||
 
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|-
 
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| Vector DNA        || ###  || ###  ||
 
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|-
 
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| 2x lgn buf (Roche) || ###  || ###  ||
 
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|-
 
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
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|-
 
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| dH<sub>2</sub>O    || ###  || ###  ||
 
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|-
 
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| &nbsp;            || # μL || # μL ||
 
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|}
 
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----
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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.
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'''Oligo annealing'''
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* Keep MV9 3-6 in case it might be useful
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# New BB 1
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# New BB 2
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
 
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|-
 
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| 10x annealing buffer || 2.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
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|}
 

Current revision

Karmella's BioBrick Cloning Main 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



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