User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/20: Difference between revisions

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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV12 = 2.3, 8.1<br>3,4. H3-1-60_MV12 = 2.3, 8.2<br>5,6. H3-1-80_MV12 = 2.3, 8.3<br>7,8. H3-1-116_MV12 = 2.3, 8.4<br>9,10. H3-1-136_MV12 = 2.3, 8.4<br>Empty MV12 = 6.9, 2.3, 1.0
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV12 = 2.3, 8.1<br>3,4. H3-1-60_MV12 = 2.3, 8.2<br>5,6. H3-1-80_MV12 = 2.3, 8.3<br>7,8. H3-1-116_MV12 = 2.3, 8.4<br>9,10. H3-1-136_MV12 = 2.3, 8.4<br>Empty MV12 = 6.9, 2.3, 1.0
| 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] = 3 μL
| rowspan="7" | [[Image:KAH102015 gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] = 3 μL
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 2.0 μL

Revision as of 20:32, 20 October 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>

10/20/15

  • Cas-tone: Digest checks on H3 constructs
  • Cas-tone: sequencing verification


Cas-tone: Digest checks on H3 constructs

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Same as 06/24/15
    • Check with SpeI/HindIII digests
    • Note: X/N H3 insert destroys SpeI site. Fragment without H3 part (Hind-Hind large) = 7960 (~8.0); add H3-1-# size + 8.0 to get length 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_MV12 = 2.3, 8.1
3,4. H3-1-60_MV12 = 2.3, 8.2
5,6. H3-1-80_MV12 = 2.3, 8.3
7,8. H3-1-116_MV12 = 2.3, 8.4
9,10. H3-1-136_MV12 = 2.3, 8.4
Empty MV12 = 6.9, 2.3, 1.0 		Hover name
15 μL/lane; 1% agarose; Ladder = 3 μL
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_MV12 a 0.194 1.885 194.084
2. H340_MV12 b 0.154 1.936 153.756
3. H360_MV12 a 0.199 1.903 199.259
4. H360_MV12 b 0.135 1.913 135.024
5. H380_MV12 a 0.179 1.931 178.795
6. H380_MV12 b 0.057 2.000 56.959
7. H3116_MV12 a 0.148 1.883 148.401
8. H3116_MV12 b 0.091 1.951 91.09
9. H3136_MV12 a 0.145 1.884 145.389
10. H3136_MV12 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-MV12-a, CMVf
  2. H340-MV12-a, DD123
  3. H340-MV12-b, CMVf
  4. H340-MV12-b, DD123
  5. H360-MV12-a, CMVf
  6. H360-MV12-a, DD123
  7. H360-MV12-b, CMVf
  8. H360-MV12-b, DD123
  9. H380-MV12-a, CMVf
  10. H380-MV12-a, DD123
  11. H3116-MV12-a, CMVf
  12. H3116-MV12-a, DD123
  13. H3116-MV12-b, CMVf
  14. H3116-MV12-b, DD123
  15. H3136-MV12-a, CMVf
  16. H3136-MV12-a, DD123
  17. H3136-MV12-b, CMVf
  18. H3136-MV12-b, DD123



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