User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22: Difference between revisions

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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV11 = 2345, 7161+##<br>3,4. H3-1-60_MV11 = 2345, 7162+##<br>5,6. H3-1-80_MV11 = 2345, 7162+##<br>7,8. H3-1-116_MV11 = 2345, 7162+##<br>9,10. H3-1-136_MV11 = 2345, 7162+##<br>
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV11 = 2345, 7161+##<br>3,4. H3-1-60_MV11 = 2345, 7162+##<br>5,6. H3-1-80_MV11 = 2345, 7162+##<br>7,8. H3-1-116_MV11 = 2345, 7162+##<br>9,10. H3-1-136_MV11 = 2345, 7162+##<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] -->
| rowspan="7" | [[Image:KAH052215_gel2.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) || 2.0 μL

Revision as of 14:44, 29 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/22/15

  • Ryan - Bra minipreps & digests
  • Cas-tone - minipreps & digests


Ryan - Bra minipreps & digests

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with EcoRI digests - promoter insertion generates a new EcoRI site
    • Note: BraR = 776, pBra = 214
Reagent Volume Expected:
1-8. pBra-BraR_MRV = 2989, 1202
empty vector = 3977
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
dH2O 10.5
  15 μL

--> 37°C/ ~15 min.


Cas-tone - H3_MV11 minipreps & digests

  • Cultures
    • Pick 2 colonies from each ligation plate
    • Make streak library & 5 mL cultures
  • Digests
    • Check with S/HindIII digests - H3 insertion destroys SpeI site
    • Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
Reagent Volume Expected:
1,2. H3-1-40_MV11 = 2345, 7161+##
3,4. H3-1-60_MV11 = 2345, 7162+##
5,6. H3-1-80_MV11 = 2345, 7162+##
7,8. H3-1-116_MV11 = 2345, 7162+##
9,10. H3-1-136_MV11 = 2345, 7162+##
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
SpeI 1.0
HindIII 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.




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