User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/09

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12/09/09

  • ✓ Cultures: KAH109-116 (2 each)
  • ✓ Assemblies: KAH117-125 (miR sensor Repressor/Target pairs)
  • ✓ Sequencing order results

Assemblies

  1. KAH117: (1) KAH99/(E/S)/2913 + (4) KAH93/(E/X)/8039
  2. KAH118: (2) KAH100/(E/S)/3085 + "
  3. KAH119: (3) KAH101/(E/S)/3157 + "
  4. KAH120: KAH99/(E/S)/2913 + (5) KAH94/(E/X)/8211
  5. KAH121: KAH100/(E/S)/3085 + "
  6. KAH122: KAH101/(E/S)/3157 + "
  7. KAH123: KAH99/(E/S)/2913 + (6) KAH95/(E/X)/8283
  8. KAH124: KAH100/(E/S)/3085 + "
  9. KAH125: KAH101/(E/S)/3157 + "

> Digests (Fermentas FD)
--> KAH99, 100, 101: E/S/ApaLI

Reagent 1-3 4-6 File:KAH 120909 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) 20.0 15.0
10x buffer 3.0 3.0
enzyme 1 1.0 1.0
enzyme 2 1.0 1.0
ApaLI 1.0 ---
dH2O 4.0 10.0
  30 μL 30 μL --> 37°C/ ~1 hr.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH99(E/S) 1.182 1.93 59.1
2. KAH100(E/S) 1.003 1.92 50.1
3. KAH101(E/S) 0.763 2.04 38.1
4. KAH93(E/X) 0.620 1.84 31.0
5. KAH94(E/X) 0.267 2.14 13.4
6. KAH95(E/X) 0.206 1.82 10.3


> Ligations

Ligation Plate results (lig : neg crtl) 12/10/09
1. KAH99(E/S)/2913, 29 ng + KAH93(E/X)/8039, 40 ng KAH117 1:1 (re-do)
2. KAH100(E/S)/3085, 31 ng + " KAH118 1:1 (re-do)
3. KAH101(E/S)/3157, 31 ng + " KAH119 1:1 (re-do)
4. KAH93(E/X)/ 40 ng  
5. KAH99(E/S)/2913, 28 ng + KAH94(E/X)/8211, 40 ng KAH120 10:1 (Pick 3)
6. KAH100(E/S)/3085, 30 ng + " KAH121 5:1 (Pick 3)
7. KAH101(E/S)/3157, 30 ng + " KAH122 2:1 (Pick 3)
8. KAH94(E/X)/ 40 ng  
9. KAH99(E/S)/2913, 28 ng + KAH95(E/X)/8283, 40 ng KAH123 3:1 (Pick 3)
10. KAH100(E/S)/3085, 30 ng + " KAH124 3:1 (Pick 3)
11. KAH101(E/S)/3157, 30 ng + " KAH125 3:1 (Pick 3)
12. KAH95(E/X)/ 40 ng  
  1 2 3 4 5 6 7 8 9 10 11 12
Insert DNA 0.5 0.6 0.8 --- 0.5 0.6 0.8 --- 0.5 0.6 0.8 ---
Vector DNA 1.3 1.3 1.3 1.3 3.0 3.0 3.0 3.0 3.9 3.9 3.9 3.9
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 6.0 6.0 6.0 6.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 2.2 2.1 1.9 2.7 0.5 0.4 0.2 1.0 0.6 0.5 0.3 1.1
  10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 12μL 12μL 12μL 12μL --> R.T./10 min.; Add 25 μL Z-DH5α

Sequencing order results

  1. KAH93/MV2 - DDB122B (fwd): good
  2. KAH93/MV2 - DDB123B (rev): good
  3. KAH94/MV2 - DDB122B (fwd): good
  4. KAH94/MV2 - DDB123B (rev): okay (1 HS4 and one partial, then non-specific sequence)
  5. KAH95/MV2 - DDB122B (fwd): good
  6. KAH95/MV2 - DDB123B (rev): good
  7. KAH99/MV2 - DDB122B (fwd): good
  8. KAH99/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
  9. KAH100/MV2 - DDB123B (fwd): good
  10. KAH100/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
  11. KAH101/MV2 - DDB122B (fwd): good
  12. KAH101/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
  13. KAH105/MV2 - DDB122B (fwd): HPK-K-Gal4DB (KAH108)? (should be Ubc-Kstop-Gal4DB)
  14. KAH105/MV2 - DDB123B (rev): good
  15. KAH106/MV2 - DDB122B (fwd): Ubc-Kstop-Gal4DB (KAH105)? (should be Ubc-K-Gal4DB); Ubc is missing a C at 21 bp
  16. KAH106/MV2 - DDB123B (rev): good
  17. KAH107/MV2 - DDB122B (fwd): Ubc-K-Gal4DB (KAH106)? (should be HPK-Kstop-Gal4DB); Ubc is missing a C at 21 bp
  18. KAH107/MV2 - DDB123B (rev): good
  19. KAH108/MV2 - DDB122B (fwd): HPK-Kstop-Gal4DB (KAH107)? (should be HPK-K-Gal4DB)
  20. KAH108/MV2 - DDB123B (rev):