User:Karmella Haynes/Notebook/BioBrick cloning/2015/08/26: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/08/26 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
Line 6: Line 6:
| 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==
==08/26/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Minipreps - CRISPR library
* Line item 2
* Cultures - CRISPR library (round 2)




----
----
'''Line item'''
'''Minipreps - CRISPR library'''
 


* Minipreps
* Minipreps
** Sigma GenElute kit, elute w/ 75 μL elution sln.
** Sigma GenElute kit, elute w/ 75 μL elution sln.
 
# L34-032
* Digests
# L34-040
** Check with #/# digests
# L34-094
 
# L34-096
{| {{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
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
 
----
'''Line item'''
 
 
* 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.
|}




Line 83: Line 34:
|}
|}


 
* Sequencing
* 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
# insert/(a/b)/size + vector/(c/d)/size
# vector/(c/d)/size
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
|-
| Insert DNA        || ###  || ---
|-
| Vector DNA        || ###  || ###
|-
| 2x lgn buf (Roche) || ###  || ###
|-
| T4 ligase (NEB)    || 1.0  || 1.0
|-
| dH<sub>2</sub>O    || ###  || ###
|-
| &nbsp;            || # μL || # μL
|}
 
 




Revision as of 16:17, 26 August 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

08/26/15

  • Minipreps - CRISPR library
  • Cultures - CRISPR library (round 2)


Minipreps - CRISPR library


  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  1. L34-032
  2. L34-040
  3. L34-094
  4. L34-096


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---
  • Sequencing



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/08/26&oldid=897618"