Haynes Lab:Notebook/Jan/2015/03/20: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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>&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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'''Plasmids - minipreps & digests'''
'''Plasmids - minipreps & digests'''
 
*'''[[User:Karmella Haynes|---Karmella]] 21:17, 26 March 2015 (EDT)''':
* Sigma GenElute Miniprep kit.
* Sigma GenElute Miniprep kit.
* Elute with 75μL elution buffer
* Elute with 75μL elution buffer
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="7" | Expected:<br>1,2. MV2 (E/X) = 4500<br>3,4. BD004 (E/S) = 4116, 3200<br>5,6. BD006 (E/S) = 4182, 3200
| rowspan="7" | Expected:<br>1,2. MV2 (E/X) = 4529*<br>3,4. BD004 (E/S) = 4116, 3200<br>5,6. BD006 (E/S) = 4182*, 3200<br>*Bands cut & purified
| rowspan="7" | [[Image:KAH032015_gel1.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH032015_gel1.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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Latest revision as of 00:50, 27 September 2017

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03/20/15

  • Plasmids - minipreps & digests


Plasmids - minipreps & digests

  • ---Karmella 21:17, 26 March 2015 (EDT):
  • Sigma GenElute Miniprep kit.
  • Elute with 75μL elution buffer
  1. MV2 a
  2. MV2 b
  3. BD004 a
  4. BD004 b
  5. BD006 a
  6. BD006 b
  • Digests (Fermentas FD)


Reagent Volume Expected:
1,2. MV2 (E/X) = 4529*
3,4. BD004 (E/S) = 4116, 3200
5,6. BD006 (E/S) = 4182*, 3200
*Bands cut & purified 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Conclusions
    • MV2 is correct
    • BD004 - most likely missing the D11.12 fragment. Sequence results from Meli'sa's past work agrees with the short fragment size on the gel.
    • BD006 is correct
  • Miniprep conc.'s
    • Combined the pairs of preps. Pairs look identical.
Sample OD260 260/280 ng/μL
1. MV2 mp 0.132 1.85 132.262
2. BD004 mp 0.144 1.91 143.967
3. BD006 mp 0.078 1.83 78.413


Assemblies

  1. BD006/MV2: BD006(E/S)/4182 + MV2(E/X)/4529


  • Gel purification: Sigma Gel extraction kit, elute and back-elute with 40 μL elution buffer
    • MV2 (E/X)
    • BD006 (E/S)


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. MV2-band1 (E/X) 0.033 0.58 32.94
2. MV2-band2 (E/X) -0.01(?) --- ---
3. BD006-band1 (E/S) -0.005(?) --- ---
4. BD006-band2 (E/S) -0.01(?) --- ---
  • Uncertain why BD006-1 yield is so low. Try using this for a ligation anyway.



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