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)
Current revision (00:42, 19 January 2013) (view source)
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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==
+
==01/10/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Violacein: successful growth; re-streak
-
* Line item 2
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* 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.)
 +
 
 +
 
 +
----
 +
'''Golden gate trial 1'''
 +
* Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
 +
* Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.
 +
 
 +
 
 +
----
 +
'''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}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
Line 20: Line 33:
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
-
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
+
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
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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] -->
+
| rowspan="7" | [[Image:KAH011013_gel1.jpg|200px|Hover name]]<br>10 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
-
| DNA(plasmid) || 2.0 μL
+
| DNA(plasmid) || 0.2 μL
|-
|-
-
| 10X buffer || 1.5
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| primer 1 || 1.0
|-
|-
-
| EcoRI || 1.0
+
| primer 2 || 1.0
|-
|-
-
| PstI || 1.0
+
| 2x GoTaq || 25.0
|-
|-
-
| dH<sub>2</sub>O || 9.5
+
| dH<sub>2</sub>O || 22.8
|-
|-
-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
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| &nbsp; || 50.0 μL
|}
|}
-
----
+
PCR
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'''Assemblies'''
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* 95°C, 3 min.
-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* 72°C, 3 min.
 +
* 4°C, ∞
-
* Digests (Fermentas FD)
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Purification
-
** Specific notes
+
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
-
 
+
* Measure [DNA]
-
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
+
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
|- valign="top"
-
| bgcolor=#cfcfcf | Reagent
+
| bgcolor=#cfcfcf | Sample
-
| bgcolor=#cfcfcf | Volume
+
| bgcolor=#cfcfcf | OD 260
-
| 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] -->
+
| bgcolor=#cfcfcf | 260/280
 +
| bgcolor=#cfcfcf | ng/μL
|-
|-
-
| DNA (plasmid) || up to 25 μL
+
| 1. pSB1A3 || 0.126 || 1.888 || 77.357
|-
|-
-
| 10x buffer || 3.0
+
| 2. hPCD || 0.069 || 2.192 || 17.025
|-
|-
-
| enzyme 1 || 1.0
+
| 3. BL01 || 0.121 || 1.898 || 66.878
-
|-
+
-
| enzyme 2 || 1.0
+
-
|-
+
-
| dH<sub>2</sub>O || ---
+
-
|-
+
-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
+
|}
|}
-
* Measure conc.'s
+
Dilutions: to final concentration of 20 fmol/μL
-
{| {{table}} cellspacing="3" <!-- [DNA] table -->
+
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
-
|- bgcolor=#cfcfcf  
+
|- valign="top"
-
| Sample || OD260 || 260/280 || ng/μL
+
| bgcolor=#cfcfcf | DNA
 +
| bgcolor=#cfcfcf | length (bp)
 +
| bgcolor=#cfcfcf | ng/μL
 +
| bgcolor=#cfcfcf | Vol.
 +
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
|-
|-
-
| 1. Digested part (a/b) || --- || --- || ---
+
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
|-
|-
-
| 2. Digested part (c/d) || --- || --- || ---
+
| hPCD || 186 || 17.0 || 2.8 || 17.2
 +
|-
 +
| BL01 || 2520 || 66.9 || 9.8 || 10.2
|}
|}
-
* Dephosphorylation (Roche)
+
Golden Gate Reactions
-
{| {{table}} cellspacing="3" <!-- Dephos table -->
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# hPCD + BL01 + pSB1A3
-
|-
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# pSB1A3
-
| 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
+
-
|}
+
-
 
+
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
-
* Ligations
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|- valign="top"
-
{| {{table}} cellspacing="3" <!-- Ligations table -->
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| Reagent || 1 || 2
-
|- 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>
+
| gg2 pSB1A3 || 1.0 || 1.0
|-
|-
-
| 2. vector(c/d)/ ## ng || &nbsp;
+
| gg3 hPCD || 1.0 || ---  
-
|}
+
-
 
+
-
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
+
-
| &nbsp;            || 1    || 2    ||
+
|-
|-
-
| Insert DNA        || ###  || --- ||
+
| gg4 BL01 || 1.0 || ---  
|-
|-
-
| Vector DNA        || ###  || ###  ||
+
| 10x ligase buffer || 1.0 || 1.0
|-
|-
-
| 2x lgn buf (Roche) || ###  || ###  ||
+
| NEB T4 lgase || 0.25 || 0.25
|-
|-
-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
+
| BsmBI || 0.5 || 0.5
|-
|-
-
| dH<sub>2</sub>O   || ###  || ###  ||
+
| dH<sub>2</sub>O || 5.25 || 7.25
|-
|-
-
| &nbsp;             || # μL || # μL ||
+
| &nbsp; || 10.0 || 10.0
|}
|}
-
----
+
Thermal cycling
-
'''Oligo annealing'''
+
* [45°C, 2 min.; 16°C, 5 min.] x25
-
# New BB 1
+
* 60°C, 20 min.
-
# New BB 2
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* 80°C, 20 min.
 +
* 4°C, ∞
-
{| 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
 
-
|}
 

Current revision

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

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
  • Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and make 2 mL liquid cultures.


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 		Hover name
10 μL/lane; 1% agarose; Ladder
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, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


Golden Gate Reactions

  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycling

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞



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