Difference between revisions of "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)
 
(01/09/13)
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| 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.|| 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
 
|}
 
  
  

Revision as of 16:57, 10 January 2013

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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.