User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09

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(Autocreate 2013/01/09 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (19:57, 10 January 2013) (view source)
(01/09/13)
 
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| colspan="2"|
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==mm/dd/yy==
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==01/09/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Golden Gate assembly: hPCD + BL01 + pSB1A3
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* Line item 2
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* Violacein: plates from Tabor; incubate o/n
----
----
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'''Minipreps'''<br>
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'''Golden Gate Assembly'''<br>
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* Check with E/P digests
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* Use purified, measured PCR products from 1/05/13
 +
* Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
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{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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* Dilutions
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{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|- valign="top"
|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| bgcolor=#cfcfcf | DNA
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| bgcolor=#cfcfcf | Volume
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| bgcolor=#cfcfcf | length (bp)
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| bgcolor=#cfcfcf | ng/μL
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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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| bgcolor=#cfcfcf | Vol.
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| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
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| DNA(plasmid) || 2.0 μL
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| gg2 pSB1A3 || 2000 || 67.7 || 7.7 || 12.3
|-
|-
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| 10X buffer || 1.5
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| gg3 hPCD || 186 || 22.5 || 2.1 || 17.8
|-
|-
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| EcoRI || 1.0
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| gg4 BL01 || 2520 || 55.9 || 11.7 || 8.3
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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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* Golden Gate Reactions
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# hPCD + BL01 + pSB1A3
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# pSB1A3
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* Digests (Fermentas FD)
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{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
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** Specific notes
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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|- valign="top"
|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| Reagent || 1 || 2
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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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| gg2 pSB1A3 || 1.0 || 1.0
|-
|-
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| 10x buffer || 3.0
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| gg3 hPCD || 1.0 || ---
|-
|-
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| enzyme 1 || 1.0
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| gg4 BL01 || 1.0 || ---
|-
|-
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| enzyme 2 || 1.0
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| 10x ligase buffer || 1.0 || 1.0
|-
|-
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| dH<sub>2</sub>O || ---
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| NEB T4 lgase || 0.25 || 0.25
|-
|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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| BsmBI || 0.5 || 0.5
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|}
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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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|-
|-
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| 1. Digested part (a/b) || --- || --- || ---
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| dH<sub>2</sub>O || 5.25 || 7.25
|-
|-
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| 2. Digested part (c/d) || --- || --- || ---
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| &nbsp; || 10.0 || 10.0
|}
|}
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Thermal cycler
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* Dephosphorylation (Roche)
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* [45°C, 2 min.; 16°C, 5 min.] x25
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{| {{table}} cellspacing="3" <!-- Dephos table -->
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* 60°C, 20 min.
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|-
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* 80°C, 20 min.
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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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|-
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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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|}
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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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|-
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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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'''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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|-
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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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-
|}
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Current revision

Karmella's BioBrick Cloning Main project page
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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.



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