Difference between revisions of "User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10"

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(Autocreate 2013/01/10 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
(mm/dd/yy)
Line 6: Line 6:
 
| colspan="2"|
 
| colspan="2"|
 
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
 
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==mm/dd/yy==
+
==01/10/13==
 
<!-- Precede finished items with a checkmark &#x2713; -->
 
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
+
* Violacein: successful growth; re-streak
* Line item 2
+
* Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
 +
* Golden gate trial 2: new PCR primers
  
  
 
----
 
----
'''Minipreps'''<br>
+
'''Violacein'''<br>
* Check with E/P digests
+
* Plate 1: Kan plasmid
 +
* Plate 2: Amp plasmid (violacein exp.)
  
{| {{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'''
+
'''Golden gate trial 1'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
+
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
  
  
* Digests (Fermentas FD)
+
----
** Specific notes
+
'''Golden gate trial 2'''
 +
* Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
  
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
+
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
|- valign="top"
 
|- valign="top"
 
| bgcolor=#cfcfcf | Reagent
 
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
+
| 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] -->
+
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
|-
+
| 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) || up to 25 μL
 
 
|-
 
|-
| 10x buffer || 3.0
+
| DNA(plasmid) || 0.2 μL
 
|-
 
|-
| enzyme 1 || 1.0
+
| primer 1 || 1.0
 
|-
 
|-
| enzyme 2 || 1.0
+
| primer 2 || 1.0
 
|-
 
|-
| dH<sub>2</sub>O || ---
+
| 2x GoTaq || 25.0
|-
 
| &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) || --- || --- || ---
+
| dH<sub>2</sub>O || 22.8
 
|-
 
|-
| 2. Digested part (c/d) || --- || --- || ---
+
| &nbsp; || 50.0 μL
 
|}
 
|}
  
 +
PCR
 +
* 95°C, 3 min.
 +
* [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
 +
* 72°C, 3 min.
 +
* 4°C, ∞
  
* 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>
 
|-
 
| 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 17:10, 10 January 2013

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01/10/13

  • Violacein: successful growth; re-streak
  • Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
  • Golden gate trial 2: new PCR primers



Violacein

  • Plate 1: Kan plasmid
  • Plate 2: Amp plasmid (violacein exp.)



Golden gate trial 1

  • Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar



Golden gate trial 2

  • Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞