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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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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.)
 
 
----
'''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 -->
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| 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
| 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
| 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.
| &nbsp; || 50.0 μL
|}
|}


----
PCR
'''Assemblies'''
* 95°C, 3 min.
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* 72°C, 3 min.
* 4°C, ∞




* Digests (Fermentas FD)
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
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
|-
| enzyme 2 || 1.0
| 1. pSB1A3 || 0.126 || 1.888 || 77.357
|-
|-
| dH<sub>2</sub>O || ---
| 2. hPCD || 0.069 || 2.192 || 17.025
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| 3. BL01 || 0.121 || 1.898 || 66.878
|}
|}




* 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
|-
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| hPCD || 186 || 17.0 || 2.8 || 17.2
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| BL01 || 2520 || 66.9 || 9.8 || 10.2
|}
|}




* Dephosphorylation (Roche)
Golden Gate Reactions
{| {{table}} cellspacing="3" <!-- Dephos table -->
# hPCD + BL01 + pSB1A3
|-
# 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
|}
 


* Ligations
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- valign="top"
|- bgcolor=#cfcfcf
| Reagent || 1 || 2
| 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
* 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
|}




Latest revision as of 22:20, 26 September 2017

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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