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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</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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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| 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" | <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>Empty MV11 = 6131, 2345, 1028
| 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]
| rowspan="7" | [[Image:KAH062415_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 2.0 μL
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CONCLUSION
CONCLUSION
* Success! Clones 1-5, 7-10 look correct.
* Success! Clones 1-5, 7-10 look correct. Clone 6 (H380_MV11 b) is empty vector
* Verify via sequencing
* Verify via sequencing


* Sequencing order (6/25/15)
* Sequencing order (6/25/15) - #10265
# H340-MV11-a, CMVf
# H340-MV11-a, CMVf
# H340-MV11-a, DD123
# H340-MV11-b, CMVf
# H340-MV11-b, DD123
# H360-MV11-a, CMVf
# H360-MV11-a, DD123
# H360-MV11-b, CMVf
# H360-MV11-b, DD123
# H380-MV11-a, CMVf
# H380-MV11-a, DD123
# H3116-MV11-a, CMVf
# H3116-MV11-a, DD123
# H3116-MV11-b, CMVf
# H3116-MV11-b, DD123
# H3136-MV11-a, CMVf
# H3136-MV11-a, DD123
# H3136-MV11-b, CMVf
# H3136-MV11-b, DD123




Latest revision as of 01:01, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

06/24/15

  • Cas-tone - H3#_MV11 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
Empty MV11 = 6131, 2345, 1028 		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.


  • DNA concentrations: BioTek Synergy H1 Take 3
Sample OD260 260/280 ng/μL
1. H340_MV11 a 0.194 1.885 194.084
2. H340_MV11 b 0.154 1.936 153.756
3. H360_MV11 a 0.199 1.903 199.259
4. H360_MV11 b 0.135 1.913 135.024
5. H380_MV11 a 0.179 1.931 178.795
6. H380_MV11 b 0.057 2.000 56.959
7. H3116_MV11 a 0.148 1.883 148.401
8. H3116_MV11 b 0.091 1.951 91.09
9. H3136_MV11 a 0.145 1.884 145.389
10. H3136_MV11 b 0.151 1.898 150.727


CONCLUSION

  • Success! Clones 1-5, 7-10 look correct. Clone 6 (H380_MV11 b) is empty vector
  • Verify via sequencing
  • Sequencing order (6/25/15) - #10265
  1. H340-MV11-a, CMVf
  2. H340-MV11-a, DD123
  3. H340-MV11-b, CMVf
  4. H340-MV11-b, DD123
  5. H360-MV11-a, CMVf
  6. H360-MV11-a, DD123
  7. H360-MV11-b, CMVf
  8. H360-MV11-b, DD123
  9. H380-MV11-a, CMVf
  10. H380-MV11-a, DD123
  11. H3116-MV11-a, CMVf
  12. H3116-MV11-a, DD123
  13. H3116-MV11-b, CMVf
  14. H3116-MV11-b, DD123
  15. H3136-MV11-a, CMVf
  16. H3136-MV11-a, DD123
  17. H3136-MV11-b, CMVf
  18. H3136-MV11-b, DD123



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