Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/19"

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(Autocreate 2013/03/19 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(03/19/13)
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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==
+
==03/19/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
+
* Oligo annealing for new mammalian vector
* Line item 2
 
  
  
----
 
'''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'''
+
'''Oligo annealing'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
* Note: made fresh 10x annealing buffer
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
 
  
* Digests (Fermentas FD)
+
# KAH3/MV2(X) + K-XbaI-NLS-his-stop
** Specific notes
+
* K-XbaI-NLS-6his-st 1
 +
* K-XbaI-NLS-6his-st 2
 +
* K-XbaI-NLS-6his-st 3
 +
* K-XbaI-NLS-6his-st 4
  
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
+
{| {{table}} <!-- Oligo annealing rxn table -->
|- valign="top"
+
| DNA (oligos, 100 μM) || 4x 3 μL ea. = 12.0
| 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 annealing buffer || 2.0
|-
 
| 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 || 6.0
 
|-
 
|-
| dH<sub>2</sub>O || ---
+
| &nbsp; || 20 μL  
|-
 
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
 
 
|}
 
|}
  
 
+
Thermal Cycling (try new thermal cycling approach)<br>
* Ligations
+
Multigene Optimax Thermal Cycler
{| {{table}} cellspacing="3" <!-- Ligations table -->
+
* 99.9 °C, 10 min.
|- bgcolor=#cfcfcf
+
* [99.9°C, temp. increment -2.0°C, 1 min.; 90.0°C, temp. increment -2.0°C, 1 min.] x35
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
+
* 25°C, ∞
|-
 
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
 
|-
 
| 2. vector(c/d)/ ## ng || &nbsp;
 
|}
 
 
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
| &nbsp;            || 1    || 2   ||
 
|-
 
| Insert DNA        || ###  || ---  ||
 
|-
 
| Vector DNA        || ###  || ###  ||
 
|-
 
| 2x lgn buf (Roche) || ###  || ###  ||
 
|-
 
| 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
 
|-
 
| dH<sub>2</sub>O || ---
 
|-
 
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
|}
 
  
  

Revision as of 13:17, 22 March 2013

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03/19/13

  • Oligo annealing for new mammalian vector



Oligo annealing

  • Note: made fresh 10x annealing buffer
  1. KAH3/MV2(X) + K-XbaI-NLS-his-stop
  • K-XbaI-NLS-6his-st 1
  • K-XbaI-NLS-6his-st 2
  • K-XbaI-NLS-6his-st 3
  • K-XbaI-NLS-6his-st 4
DNA (oligos, 100 μM) 4x 3 μL ea. = 12.0
10x annealing buffer 2.0
dH2O 6.0
  20 μL

Thermal Cycling (try new thermal cycling approach)
Multigene Optimax Thermal Cycler

  • 99.9 °C, 10 min.
  • [99.9°C, temp. increment -2.0°C, 1 min.; 90.0°C, temp. increment -2.0°C, 1 min.] x35
  • 25°C, ∞