User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base: Difference between revisions
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{|{{table}} width="800" | {|{{table}} width="800" | ||
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |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> </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"|<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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
|- | |- | ||
| 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== | ||
* Insert | <!-- Precede finished items with a checkmark ✓ --> | ||
* Line item 1 | |||
* 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 | |||
|- | |||
| || 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 || --- | |||
|- | |||
| || 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 || --- | |||
|- | |||
| || 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 || | |||
|} | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 || 2 || | |||
|- | |||
| Insert DNA || ### || --- || | |||
|- | |||
| Vector DNA || ### || ### || | |||
|- | |||
| 2x lgn buf (Roche) || ### || ### || | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || | |||
|- | |||
| dH<sub>2</sub>O || ### || ### || | |||
|- | |||
| || # 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 || --- | |||
|- | |||
| || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight | |||
|} | |||
Revision as of 14:35, 1 December 2012
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
mm/dd/yy
Minipreps
Assemblies
Oligo annealing
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