User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(13 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|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== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Line item 1 | * Line item 1 | ||
* Line item 2 | * Line item 2 | ||
---- | ---- | ||
'''Minipreps'''<br> | '''Minipreps'''<br> | ||
* Check with E/P digests | |||
{| | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|-valign="top" | |- 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" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br> | ||
| rowspan="7" | <!-- | | 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 | | DNA(plasmid) || 2.0 μL | ||
Line 39: | Line 41: | ||
# 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) | ||
|-valign="top" | ** Specific notes | ||
| | |||
| rowspan=" | {| {{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 | | DNA (plasmid) || up to 25 μL | ||
Line 62: | Line 65: | ||
* Measure conc.'s | |||
{| | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- | |- bgcolor=#cfcfcf | ||
| | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. Digested part (a/b) || --- || --- || --- | | 1. Digested part (a/b) || --- || --- || --- | ||
Line 73: | Line 76: | ||
* Dephosphorylation (Roche) | |||
{| | {| {{table}} cellspacing="3" <!-- Dephos table --> | ||
|- | |- | ||
| | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | |||
|- | |- | ||
| DNA (clean digest) || up to 17 μL (500 ng) | | DNA (clean digest) || up to 17 μL (500 ng) | ||
Line 90: | Line 94: | ||
* 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. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font> | ||
|- | |- | ||
| 2. vector(c/d)/ ## ng || | | 2. vector(c/d)/ ## ng || | ||
|} | |} | ||
{| | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || | ||
|- | |- | ||
| Insert DNA || ### || --- || | | Insert DNA || ### || --- || | ||
|- | |- | ||
| Vector DNA || ### || ### || | | Vector DNA || ### || ### || | ||
|- | |- | ||
| 2x lgn buf (Roche) || ### || ### || | | 2x lgn buf (Roche) || ### || ### || | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || | ||
|- | |- | ||
| dH<sub>2</sub>O || ### || ### || | | dH<sub>2</sub>O || ### || ### || | ||
|- | |- | ||
| || # μL || # μL || | | || # μL || # μL || |
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
|