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
|
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
- 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
|
Karmella's BioBrick Cloning
Main project page
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