User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15

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* Make Promega buffer from scratch. Test Promega and NEB formulas.
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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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| 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 || ---
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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* Measure conc.'s
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
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|- bgcolor=#cfcfcf
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| Sample || OD260 || 260/280 || ng/μL
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| 1. Digested part (a/b) || --- || --- || ---
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| 2. Digested part (c/d) || --- || --- || ---
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* Dephosphorylation (Roche)
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{| {{table}} cellspacing="3" <!-- Dephos table -->
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | Volume
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| DNA (clean digest) || up to 17 μL (500 ng)
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| 10x buffer d.p. || 2.0
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| phosphatase || 1.0
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| dH<sub>2</sub>O || ---
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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* Ligations
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{| {{table}} cellspacing="3" <!-- Ligations table -->
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|- bgcolor=#cfcfcf
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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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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| 2. vector(c/d)/ ## ng || &nbsp;
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    ||
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| 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  ||
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| dH<sub>2</sub>O    || ###  || ###  ||
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| &nbsp;            || # μL || # μL ||
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----
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'''Oligo annealing'''
 
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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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| 10x annealing buffer || 2.0
 
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| dH<sub>2</sub>O || ---
 
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
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Revision as of 18:48, 15 January 2013

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

01/15/13

  • Golden gate assembly: test different ligase buffers



Golden Gate Assembly optimization

  • Test different buffers
Buffer Tris-HCl MgCl2 DTT ATP
10x Promega 300 mM (pH 7.8) 100 mM 100 mM 10 mM
10x NEB 500 mM (pH 7.5) 100 mM 100 mM 10 mM
2x Roche  ?  ?  ?  ?
  • Make Promega buffer from scratch. Test Promega and NEB formulas.


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