User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/21

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(Autocreate 2013/03/21 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (22:01, 24 March 2013) (view source)
(→03/21/13)
 
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==mm/dd/yy==
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==03/21/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Assemblies: retry MV9
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* Line item 2
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* T4 ligase quality check: Behzad's tube
-
 
-
----
 
-
'''Minipreps'''<br>
 
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* Check with E/P digests
 
-
 
-
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
-
|- 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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| DNA(plasmid) || 2.0 μL
 
-
|-
 
-
| 10X buffer || 1.5
 
-
|-
 
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| EcoRI || 1.0
 
-
|-
 
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| PstI || 1.0
 
-
|-
 
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| dH<sub>2</sub>O || 9.5
 
-
|-
 
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
 
-
|}
 
----
----
'''Assemblies'''
'''Assemblies'''
-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
* Try again, but with the old boil and cool method
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
-
* Digests (Fermentas FD)
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* <font color="blue">'''Oligo annealing'''</font>
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** Specific notes
+
* See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/19 03/19/13] for details about the oligos and reaction set-up
 +
** Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).
 +
** Float the oligo mixtures in the boiling water for 10 min. If there is condensation, carefully & quickly remove the tube and shoe the liquid down to the bottom and put the tubes back into the water.
 +
** Turn off the heat source and allow the water bath to slowly cool to room temperature (25°C) for several hours.
-
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 
-
|- valign="top"
 
-
| bgcolor=#cfcfcf | Reagent
 
-
| 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] -->
 
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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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| dH<sub>2</sub>O || ---
 
-
|-
 
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
-
|}
 
 +
* '''Digest''' (Fermentas FD) - MV2 vector, [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13]
 +
* '''Measure conc.''' -  [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13]
 +
* '''Dephosphorylation''' (Roche) - MV2 XbaI-cut vector,  [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20 03/20/13]
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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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* '''Ligations'''
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* Dephosphorylation (Roche)
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{| {{table}} cellspacing="3" <!-- Ligations table -->
-
{| {{table}} cellspacing="3" <!-- Dephos table -->
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|- bgcolor=#cfcfcf
 +
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/22/13</font>
|-
|-
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| bgcolor=#cfcfcf | Reagent
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| 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 lawn</font>
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| bgcolor=#cfcfcf | Volume
+
|-
|-
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| DNA (clean digest) || up to 17 μL (500 ng)
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| 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 lawn</font>
|-
|-
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| 10x buffer d.p. || 2.0
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| 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="red">MV9 lawn</font>
|-
|-
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| phosphatase || 1.0
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| 4. MV9(X dp)/ 25 ng || &nbsp;
|-
|-
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| dH<sub>2</sub>O || ---
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| 5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase || <font color="red">lawn</font>
|-
|-
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
+
| 6. MV9(X no dp)/ 25 ng || &nbsp;
|}
|}
-
 
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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;
 
-
|}
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2    ||
+
| &nbsp;            || 1    || 2    || 3    || 4  ||  9  || 10 
|-
|-
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| Insert DNA        || ### || ---  ||
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| Insert DNA        || 0.5  || 1.0  || 2.0 || ---  || ---  || --- 
|-
|-
-
| Vector DNA        || ### || ### ||
+
| Vector DNA        || 1.0 || 1.0 || 1.0  || 1.0  || 0.3  || 0.3
|-
|-
-
| 2x lgn buf (Roche) || ### || ### ||
+
| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.0  || 5.0  || 5.0  || 5.0
|-
|-
-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
+
| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0  || 1.0  || 1.0  || ---
|-
|-
-
| dH<sub>2</sub>O    || ### || ### ||
+
| dH<sub>2</sub>O    || 2.5 || 2.0 || 1.0  || 3.0  || 3.7  || 4.7
|-
|-
-
| &nbsp;            || # μL || # μL ||
+
| &nbsp;            || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL
|}
|}
-
----
+
* <font color="blue">Pre-warm agar-amp plates with lids closed</font>
-
'''Oligo annealing'''
+
* <font color="blue">'''30 min.'''/ room temp</font>
-
# New BB 1
+
* Add 30 μL DH5α; 5 min/ ice
-
# New BB 2
+
* Plate on 100 μg/mL amp; incubate at 37°C
-
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
-
| 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
 
-
|}
 
 +
3/22/13<br>
 +
* Plates have bacterial lawns. Used plates from Behzad's "LB Plane Agar" sleeve because it was marked with red tape and nobody, conveniently, reported that we ran out of LB amp. Thanks, guys.
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Current revision

Karmella's BioBrick Cloning Main project page
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03/21/13

  • Assemblies: retry MV9
  • T4 ligase quality check: Behzad's tube


Assemblies

  • Try again, but with the old boil and cool method


  • Oligo annealing
  • See 03/19/13 for details about the oligos and reaction set-up
    • Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).
    • Float the oligo mixtures in the boiling water for 10 min. If there is condensation, carefully & quickly remove the tube and shoe the liquid down to the bottom and put the tubes back into the water.
    • Turn off the heat source and allow the water bath to slowly cool to room temperature (25°C) for several hours.


  • Digest (Fermentas FD) - MV2 vector, 03/20/13
  • Measure conc. - 03/20/13
  • Dephosphorylation (Roche) - MV2 XbaI-cut vector, 03/20/13


  • Ligations
Ligation Plate results (lig : neg crtl) 03/22/13
1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 lawn
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 lawn
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 lawn
4. MV9(X dp)/ 25 ng  
5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase lawn
6. MV9(X no dp)/ 25 ng  


  1 2 3 4 9 10
Insert DNA 0.5 1.0 2.0 --- --- ---
Vector DNA 1.0 1.0 1.0 1.0 0.3 0.3
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 ---
dH2O 2.5 2.0 1.0 3.0 3.7 4.7
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL
  • Pre-warm agar-amp plates with lids closed
  • 30 min./ room temp
  • Add 30 μL DH5α; 5 min/ ice
  • Plate on 100 μg/mL amp; incubate at 37°C


3/22/13

  • Plates have bacterial lawns. Used plates from Behzad's "LB Plane Agar" sleeve because it was marked with red tape and nobody, conveniently, reported that we ran out of LB amp. Thanks, guys.


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