Haynes Lab:Notebook/Jan/2015/03/26: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|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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==Entry title==
==03/26/15==
* Insert content here...
* Plasmid miniprep - (2x) 5 mL liquid culture for DPRE (D11.12)
* Assembly - BD006 into MV2
 
 
----
'''Assembly'''
*'''[[User:Karmella Haynes|---Karmella]] 21:16, 26 March 2015 (EDT)''':
* BD006/MV2: insert BD006(E/S)/4182 + vector MV2(E/X)/4529
* Use Roche 2x buffer and NEB T4 ligase
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/20
 
 
* Ligations
# 10 uL rxn, vector + insert
# 10 uL rxn, vector
# 15 uL rxn, vector + insert
# 15 uL rxn, vector
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | 1
| bgcolor=#cfcfcf | 2
| bgcolor=#cfcfcf | 3
| bgcolor=#cfcfcf | 4
|-
| DNA insert || 3.0 || --- || 5.5 || ---
|-
| DNA vector || 1.5 || 1.5 || 1.5 || 1.5
|-
| 2x buffer || 5.0 || 5.0 || 7.5 || 7.5
|-
| T4 ligase || 0.5 || 0.5 || 0.5 || 0.5
|-
| dH<sub>2</sub>O || --- || 3.0 || --- || 5.5
|-
| &nbsp; || 10 μL  || 10 μL  || 15 μL  || 15 μL
|}
 
--> RT/ ~30 min.
* Add 50 μL DH5-α turbo
* 5 min./ ice
* Plate on 100 ug/mL amp agar. Grow at 37 °C o/n
 
 
'''RESULTS''' (3/27/15)
# BD006/MV2, 10 uL rxn, vector + insert: 6 colonies
# BD006/MV2,10 uL rxn, vector: zero
# BD006/MV2,15 uL rxn, vector + insert: 4 colonies
# BD006/MV2,15 uL rxn, vector: zero
 
* Success! Pick two colonies from each plate (1 and 3) for minipreps
 
 
 




Latest revision as of 00:52, 27 September 2017

Project name Main project page
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03/26/15

  • Plasmid miniprep - (2x) 5 mL liquid culture for DPRE (D11.12)
  • Assembly - BD006 into MV2


Assembly


  • Ligations
  1. 10 uL rxn, vector + insert
  2. 10 uL rxn, vector
  3. 15 uL rxn, vector + insert
  4. 15 uL rxn, vector
Reagent 1 2 3 4
DNA insert 3.0 --- 5.5 ---
DNA vector 1.5 1.5 1.5 1.5
2x buffer 5.0 5.0 7.5 7.5
T4 ligase 0.5 0.5 0.5 0.5
dH2O --- 3.0 --- 5.5
  10 μL 10 μL 15 μL 15 μL

--> RT/ ~30 min.

  • Add 50 μL DH5-α turbo
  • 5 min./ ice
  • Plate on 100 ug/mL amp agar. Grow at 37 °C o/n


RESULTS (3/27/15)

  1. BD006/MV2, 10 uL rxn, vector + insert: 6 colonies
  2. BD006/MV2,10 uL rxn, vector: zero
  3. BD006/MV2,15 uL rxn, vector + insert: 4 colonies
  4. BD006/MV2,15 uL rxn, vector: zero
  • Success! Pick two colonies from each plate (1 and 3) for minipreps




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