User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/24: Difference between revisions

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'''Cas-tone - minipreps, digests'''
'''Cas-tone - minipreps, digests'''


* Minipreps
'''Cas-tone - H3_MV11 minipreps & digests'''
** Sigma GenElute kit, elute w/ 75 μL elution sln.
 
* Cultures
** Pick 2 colonies from each ligation plate
** Make streak library & 5 mL cultures


* Digests
* Digests
** Check with #/# digests
** Sigma GenElute kit; elute w/ 75μL eln. buffer
** 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
** H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433


{| {{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><br>1,2. H3-1-40_MV11 = 2345, 7291<br>3,4. H3-1-60_MV11 = 2345, 7351<br>5,6. H3-1-80_MV11 = 2345, 7412<br>7,8. H3-1-116_MV11 = 2345, 7535<br>9,10. H3-1-136_MV11 = 2345, 7595<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
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| 10X buffer || 1.5
| 10X buffer || 1.5
|-
|-
| EcoRI || 1.0
| SpeI || 1.0
|-
|-
| PstI || 1.0
| HindIII || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 9.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL  
|}
|}
 
--> 37°C/ ~15 min.
 




Revision as of 18:47, 24 June 2015

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06/24/15

  • Cas-tone - minipreps, digests


Cas-tone - minipreps, digests

Cas-tone - H3_MV11 minipreps & digests

  • Cultures
    • Pick 2 colonies from each ligation plate
    • Make streak library & 5 mL cultures
  • Digests
    • Sigma GenElute kit; elute w/ 75μL eln. buffer
    • 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
    • H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433
Reagent Volume Expected:
1,2. H3-1-40_MV11 = 2345, 7291
3,4. H3-1-60_MV11 = 2345, 7351
5,6. H3-1-80_MV11 = 2345, 7412
7,8. H3-1-116_MV11 = 2345, 7535
9,10. H3-1-136_MV11 = 2345, 7595
 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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