User:Karmella Haynes/Notebook/BioBrick cloning/2015/08/27: Difference between revisions

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(Autocreate 2015/08/27 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==08/27/15==
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* Line item 1
* Minipreps- CRISPR library cultures (round 2)
* Line item 2
* Line item 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.
 
# G34-024
* Digests
# G34-067
** Check with #/# digests
# G34-079
 
{| {{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.
|}




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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. G34-024 || 0.167 || 1.863 || 167.3
|-
| 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 || ---
| 2. G34-067 || 0.026 || 1.908 || 26.4
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| 3. G34-079 || 0.032 || 1.901 || 32.4
|}
|}


 
* Sequencing: Order 10437
* Ligations
# G34-024, P163
# insert/(a/b)/size + vector/(c/d)/size
# G34-067 "
# vector/(c/d)/size
# G34-079 "
 
{| {{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 14:58, 27 August 2015

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08/27/15

  • Minipreps- CRISPR library cultures (round 2)
  • Line item 2


Minipreps - CRISPR library

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  1. G34-024
  2. G34-067
  3. G34-079


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. G34-024 0.167 1.863 167.3
2. G34-067 0.026 1.908 26.4
3. G34-079 0.032 1.901 32.4
  • Sequencing: Order 10437
  1. G34-024, P163
  2. G34-067 "
  3. G34-079 "




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