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

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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
| 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] -->
| 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]
|-
|-
| DNA (plasmid) || 20.0 μL
| DNA (plasmid) || 20.0 μL
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** 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.
** 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
** BD006  is correct
* Miniprep conc.'s
** Combined the pairs of preps. Pairs look identical.
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| 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'''
'''Assemblies'''
# BD006/MV2: BD006(E/S)/4000 + MV2(E/X)/4500
# BD006/MV2: BD006(E/S)/4182 + MV2(E/X)/4529




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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. MV2 (E/X) || --- || --- || ---
| 1. MV2-band1 (E/X) || 0.033 || 0.58 || 32.94
|-
|-
| 2. MV2 (E/X) || --- || --- || ---
| 2. MV2-band2 (E/X) || -0.01(?) || --- || ---
|-
|-
| 3. BD006 (E/S) || --- || --- || ---
| 3. BD006-band1 (E/S) || -0.005(?) || --- || ---
|-
|-
| 4. BD006 (E/S) || --- || --- || ---
| 4. BD006-band2 (E/S) || -0.01(?) || --- || ---
|}
|}


* Uncertain why BD006-1 yield is so low. Try using this for a ligation anyway.




Revision as of 18:17, 26 March 2015

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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 		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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