User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05

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(Autocreate 2013/01/05 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
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==mm/dd/yy==
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==01/05/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Golden gate: PCR parts, complete protocol for Brady's construct
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* Line item 2
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* Gibson: PCR parts, complete protocol for Brady's construct
----
----
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'''Minipreps'''<br>
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'''Golden Gate Assembly'''<br>
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* Check with E/P digests
+
* Try new protocol from Dave Savage
 +
# hPCD + BL01 + V0120
 +
# hPCD + BL01 + pSB1A3
 +
 
 +
* Check the sequences for BsmBI sites
 +
 
 +
'''PCR'''
 +
# V0120, gg_? / gg_?
 +
# pSB1A3, gg_? / gg_?
 +
# hPCD, gg_BBP-hPCD F  / gg_hPCD R
 +
# BL01, gg_BL01 F / gg_BL01-BBS R
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| 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" | <u>Expected:</u><br>1. V0120 = size<br>2. pSB1A3 = size<br>3. hPCD = size<br>4. BL01 = size
| 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] -->
| 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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| DNA(plasmid) || 0.5 μL
|-
|-
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| 10X buffer || 1.5
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| 10 μM primer 1 || 1.0
|-
|-
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| EcoRI || 1.0
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| 10 μM primer 2 || 1.0
|-
|-
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| PstI || 1.0
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| 2x GoTaq mix || 25.0
|-
|-
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| dH<sub>2</sub>O || 9.5
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| dH<sub>2</sub>O || ##
|-
|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
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| &nbsp; || 50 μL  
|}
|}
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----
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* 95°C, 3 min.
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'''Assemblies'''
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* 72°C, 3 min.
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* 4°C, ∞
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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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| 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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-
 
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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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| 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
 
-
|-
 
-
| 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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|-
 
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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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'''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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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
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|}
 

Revision as of 14:30, 5 January 2013

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

01/05/13

  • Golden gate: PCR parts, complete protocol for Brady's construct
  • Gibson: PCR parts, complete protocol for Brady's construct


Golden Gate Assembly

  • Try new protocol from Dave Savage
  1. hPCD + BL01 + V0120
  2. hPCD + BL01 + pSB1A3
  • Check the sequences for BsmBI sites

PCR

  1. V0120, gg_? / gg_?
  2. pSB1A3, gg_? / gg_?
  3. hPCD, gg_BBP-hPCD F / gg_hPCD R
  4. BL01, gg_BL01 F / gg_BL01-BBS R
Reagent Volume Expected:
1. V0120 = size
2. pSB1A3 = size
3. hPCD = size
4. BL01 = size
DNA(plasmid) 0.5 μL
10 μM primer 1 1.0
10 μM primer 2 1.0
2x GoTaq mix 25.0
dH2O ##
  50 μL
  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
  • 72°C, 3 min.
  • 4°C, ∞




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