User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/06: Difference between revisions

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----
----
'''Minipreps'''<br>
'''Minipreps'''<br>
* Check with KpnI digests
* Use Sigma GenElute; elute with 75 μL elution solution
* '''promoter/Regulator/MRV colonies'''
* '''Cut w/ KpnI/EcoRI''' - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
# pAub-AubR/MRV
# pBja-BjaR/MRV
# pBra-BraR/MRV
# pRpa-RpaR/MRV
 


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| rowspan="7" | <u>Expected:</u>1,2. pAub/AubR/MRV = 4240<br>3,4. pBja/BjaR/MRV = 4099<br>5,6. pBra/BraR/MRV = ###<br>7,8. pRpa/RpaR/MRV = 4116<br>Empty vector = ~4000, 971
| 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] -->
| rowspan="7" | [[Image:KAH042815_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 3.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 1.5
|-
|-
| EcoRI || 1.0
| enzyme 1 || 1.0
|-
|-
| PstI || 1.0
| enzyme 2 || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 8.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
|}


* Measure [DNA]
{| {{table}}
|-
| Sample || OD 260 || 260/280 || ng/μL
|-
| 1. || 0.465 || 1.913 || 465.4
|}
Conclusions
* tba




Revision as of 18:39, 6 May 2015

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05/05/15

  • Ryan - minipreps & digests


Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • promoter/Regulator/MRV colonies
  • Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
  1. pAub-AubR/MRV
  2. pBja-BjaR/MRV
  3. pBra-BraR/MRV
  4. pRpa-RpaR/MRV


Reagent Volume Expected:1,2. pAub/AubR/MRV = 4240
3,4. pBja/BjaR/MRV = 4099
5,6. pBra/BraR/MRV = ###
7,8. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. 0.465 1.913 465.4


Conclusions

  • tba



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