User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/21: Difference between revisions

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(Autocreate 2013/03/21 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==mm/dd/yy==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Assemblies: retry MV9
* Line item 2
* T4 ligase quality check: Behzad's tube


----
'''Minipreps'''<br>
* Check with E/P digests
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &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
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
* '''Oligo annealing'''
** 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] -->
|-
| 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.
|}


* '''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]


* 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) || --- || --- || ---
|}


 
* '''Ligations'''
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Ligations table -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font>
|-
|-
| bgcolor=#cfcfcf | Reagent
| 1. A1 (ds oligo S/S)/76, ''0.5 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font>
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| 2. A1 (ds oligo S/S)/76, ''1.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font>
|-
|-
| 10x buffer d.p. || 2.0
| 3. A1 (ds oligo S/S)/76, ''2.0 μL'' + MV2(X dp)/4529, 25 ng || <font color="blue">MV9 ##:##</font>
|-
|-
| phosphatase || 1.0
| 4. MV9(X dp)/ 25 ng || &nbsp;
|-
| 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>
| 5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase || <font color="blue">...</font>
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| 6. MV9(X no dp)/ 25 ng || &nbsp;
|}
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    || 3    || 4  ||  9  || 10 
|-
|-
| Insert DNA        || ### || ---  ||
| 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  ||
|-
| dH<sub>2</sub>O    || ### || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}
 
----
'''Oligo annealing'''
# New BB 1
# New BB 2
 
{| 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
| 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; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
| &nbsp;             || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL || 10 μL  
|}
|}


* 10 min./ room temp
* Add 30 μL DH5α; 5 min/ ice
* Plate on 100 μg/mL amp; incubate at 37°C


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 19:48, 21 March 2013

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mm/dd/yy

  • 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) 03/21/13
1. A1 (ds oligo S/S)/76, 0.5 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
2. A1 (ds oligo S/S)/76, 1.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
3. A1 (ds oligo S/S)/76, 2.0 μL + MV2(X dp)/4529, 25 ng MV9 ##:##
4. MV9(X dp)/ 25 ng  
5. MV9(X no dp)/ 25 ng + Behzad's T4 ligase ...
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
  • 10 min./ room temp
  • Add 30 μL DH5α; 5 min/ ice
  • Plate on 100 μg/mL amp; incubate at 37°C


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