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
|
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 |
0.07 |
1.812 |
70.263
|
| 2. H340_MV12 b |
0.06 |
1.915 |
59.669
|
| 3. H360_MV12 a |
0.072 |
1.882 |
71.57
|
| 4. H360_MV12 b |
0.052 |
1.925 |
51.593
|
| 5. H380_MV12 a |
0.072 |
1.818 |
72.02
|
| 6. H380_MV12 b |
0.029 |
1.814 |
28.636
|
| 7. H3116_MV12 a |
0.037 |
1.865 |
36.735
|
| 8. H3116_MV12 b |
0.056 |
1.844 |
56.147
|
| 9. H3136_MV12 a |
0.071 |
1.84 |
70.707
|
| 10. H3136_MV12 b |
0.041 |
1.926 |
40.657
|
CONCLUSION
- Success! Clones 1-10 look correct.
- Verify via sequencing
- Sequencing order (10/21/15) - #
- H340-MV12-a, CMVf
- H340-MV12-a, DD123
- H340-MV12-b, CMVf
- H340-MV12-b, DD123
- H360-MV12-a, CMVf
- H360-MV12-a, DD123
- H360-MV12-b, CMVf
- H360-MV12-b, DD123
- H380-MV12-a, CMVf
- H380-MV12-a, DD123
- H380-MV12-b, CMVf
- H380-MV12-b, DD123
- H3116-MV12-a, CMVf
- H3116-MV12-a, DD123
- H3116-MV12-b, CMVf
- H3116-MV12-b, DD123
- H3136-MV12-a, CMVf
- H3136-MV12-a, DD123
- H3136-MV12-b, CMVf
- H3136-MV12-b, DD123
|
Karmella's BioBrick Cloning
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