User:Karmella Haynes/Notebook/BioBrick cloning/2015/03/26: 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==
==03/26/15==
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* '''PEG precipitation - optimization'''
* '''PEG precipitation - optimization'''
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----
----
'''PEG precipitation - optimization'''<br>
'''PEG precipitation - optimization'''<br>
* Make PEG
* See OWW protocol - [http://openwetware.org/wiki/Protocol_Size_selective_DNA_precipitation_by_PEG/MgCl2 Size selective DNA precipitation]
* In this procedure, PEG is diluted 3-fold in the final DNA mixture
* Try these final PEG concentrations: 10%, 6%, 5%, 4%, 3%, 2%
 
 
'''Make PEG solutions'''
* Make TE Buffer, pH 8.0: 10 mM TRIS-HCl, 0.1 mM EDTA, pH 8.0
* Make 30 mM MgCl2, 50 mL (plus one extra): 1.5 mL 1M MgCl2 + 48.5 molecular bio grade H<sub>2</sub>O
* Make 30% PEG, 50 mL: 1.5 g PEG 8000 + 50 mL 30 mM MgCL2
* Use this to make other solutions


{| {{table}} border="1" cellspacing="3" <!-- Digest check 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 | 18% PEG 
| bgcolor=#cfcfcf | 15% PEG 
| bgcolor=#cfcfcf | 12% PEG
| bgcolor=#cfcfcf | 9% PEG   
| bgcolor=#cfcfcf | 6% PEG   
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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] -->
| 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
| 30% PEG || 6.0 mL || 5.0 mL || 4.0 mL || 3.0 mL || 2.0 mL
|-
| 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'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
 
 
* Digests (Fermentas FD)
** Specific notes
 
{| {{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.
|}
 
 
* 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 || ---
|-
| &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        || ###  || ---  ||
| 30 mM MgCl2 || 4.0 mL || 5.0 mL || 6.0 mL || 7.0 mL || 8.0 mL
|-
|-
| Vector DNA        || ###  || ###  ||
| &nbsp; || 10 mL
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;             || # μL || # μL ||
|}
|}


----
'''Procedure'''
'''Oligo annealing'''
* Make 6 samples of DNA ladder (1 kb Plus): 1 μL (500 ng) + 49 μL H<sub>2</sub>O
# New BB 1
* Mix 50 μL of sample with 150 µL of TE
# New BB 2
* Add 100 µL of PEG/MgCl2
* Vortex
* Centrifuge 15 min at 10,000 rcf at room temperature. Note: orient the tubes "hinge" side up so that the onvisible pellet will be located towards the hinge (at bottom of tube).
* Carefully transfer ALL supernatant to a new tube. Do not to disturb the pellet, which will be invisible
* Dissolve the pellet in 50 μL dH<sub>2</sub>O


{| 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 00:52, 27 September 2017

Karmella's BioBrick Cloning Main project page
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03/26/15

  • PEG precipitation - optimization


PEG precipitation - optimization

  • See OWW protocol - Size selective DNA precipitation
  • In this procedure, PEG is diluted 3-fold in the final DNA mixture
  • Try these final PEG concentrations: 10%, 6%, 5%, 4%, 3%, 2%


Make PEG solutions

  • Make TE Buffer, pH 8.0: 10 mM TRIS-HCl, 0.1 mM EDTA, pH 8.0
  • Make 30 mM MgCl2, 50 mL (plus one extra): 1.5 mL 1M MgCl2 + 48.5 molecular bio grade H2O
  • Make 30% PEG, 50 mL: 1.5 g PEG 8000 + 50 mL 30 mM MgCL2
  • Use this to make other solutions
Reagent 18% PEG 15% PEG 12% PEG 9% PEG 6% PEG Expected:
1. BB 1 = size
2. BB2 = size
30% PEG 6.0 mL 5.0 mL 4.0 mL 3.0 mL 2.0 mL
30 mM MgCl2 4.0 mL 5.0 mL 6.0 mL 7.0 mL 8.0 mL
  10 mL

Procedure

  • Make 6 samples of DNA ladder (1 kb Plus): 1 μL (500 ng) + 49 μL H2O
  • Mix 50 μL of sample with 150 µL of TE
  • Add 100 µL of PEG/MgCl2
  • Vortex
  • Centrifuge 15 min at 10,000 rcf at room temperature. Note: orient the tubes "hinge" side up so that the onvisible pellet will be located towards the hinge (at bottom of tube).
  • Carefully transfer ALL supernatant to a new tube. Do not to disturb the pellet, which will be invisible
  • Dissolve the pellet in 50 μL dH2O



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