User:Robwarden/Notebook/Divalent Ligand Validation/Revised Cloning Strategy
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PCR Amplification
Her1/EGFR
Primer Design
- Sense Primer
- 5'-gacttacg(Spacer)-ctcgag(XhoI)-atgcgaccctccgggacggc(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 68°C (Annealing), 74°C Overall
- 75% GC (Annealing), 68% Overall
- Antisense Primer
- 5'-gacttcag(Spacer)-gcggccgc(NotI)-tcatta(Stop)-GGAACCTCCCCCGCCGCTACCGCCGCCCCCACGCGTACCCCCTCCGCC(Linker w/ MluI)-caatgctccaataaattcactgctttgtggc(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 63°C (Annealing), 85°C Overall
- 42% GC (Annealing, 66% Overall
Reaction Cycle
| Cycles | Temp (°C) | Time |
|---|---|---|
| 1 | 98 | 0:30 |
| 35 | 98 | 0:10 |
| 66 | 0:30 | |
| 72 | 2:00 | |
| 1 | 72 | 5:00 |
| 4 | ∞ |
Her3
Primer Design
- Sense Primer
- 5'-gacttcag(Spacer)-gtcgac(SalI)-atgagggcgaacgacgctctg(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 63°C (Annealing), 71°C Overall
- 62% GC (Annealing), 60% Overall
- Antisense Primer
- 5'-gacttcag(Spacer)-gcggccgc(NotI)-tcatta(Stop)-GGAACCTCCCCCGCCGCTACCGCCGCCCCCACGCGTACCCCCTCCGCC(Linker w/ MluI)-cgttctctgggcattagccttggg(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 63°C (Annealing), 88°C Overall
- 58% GC (Annealing, 74% Overall
Reaction Cycle
| Cycles | Temp (°C) | Time |
|---|---|---|
| 1 | 98 | 0:30 |
| 35 | 98 | 0:10 |
| 66 | 0:30 | |
| 72 | 2:00 | |
| 1 | 72 | 5:00 |
| 4 | ∞ |
CyPet
Primer Design
- Sense Primer
- 5'-GGCGGAGGGGGTACGCGTGGGGGCGGCGGTAGCGGCGGGGGAGGTTCC(Linker w/ MluI)-atggtgagcaagggagaggaactg(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 62°C (Annealing), 87°C Overall
- 54% GC (Annealing), 72% Overall
- Antisense Primer
- 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttatttgtacagttcgtccatgccgtgg(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 63°C (Annealing), 75°C Overall
- 46% GC (Annealing, 57% Overall
Reaction Cycle
| Cycles | Temp (°C) | Time |
|---|---|---|
| 1 | 98 | 0:30 |
| 35 | 98 | 0:10 |
| 65 | 0:30 | |
| 72 | 0:30 | |
| 1 | 72 | 5:00 |
| 4 | ∞ |
YPet
Primer Design
- Sense Primer
- 5'-GGCGGAGGGGGTACGCGTGGGGGCGGCGGTAGCGGCGGGGGAGGTTCC(Linker w/ MluI)-atggtgagcaaaggcgaagagc(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 62°C (Annealing), 87°C Overall
- 55% GC (Annealing), 73% Overall
- Antisense Primer
- 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttacttatagagctcgttcatgccctcg(Annealing)-3'
- [math]\displaystyle{ T_m }[/math] = 62°C (Annealing), 74°C Overall
- 46% GC (Annealing, 57% Overall
Reaction Cycle
| Cycles | Temp (°C) | Time |
|---|---|---|
| 1 | 98 | 0:30 |
| 35 | 98 | 0:10 |
| 65 | 0:30 | |
| 72 | 0:30 | |
| 1 | 72 | 5:00 |
| 4 | ∞ |
ErbB Insertion
Restriction Digests
Her1/EGFR
- NotI & XhoI
- Digest in NEBuffer 3 + BSA at 37°C.
Her3
- NotI & SalI
- Digest in NEBuffer 3 + BSA at 37°C.
pMSCVN
- NotI & XhoI
- Digest in NEBuffer 3 + BSA at 37°C.
Ligation
As per NEB Quick Ligase Protocol
Transformation
- Into XL1-Blue Competent Cells
- AmpR
Validation
pMSCVN-Her1
- EcoRI Digest
- Good: 6581, 1838, 797, 768bp
- Empty: 6303bp
pMSCVN-Her3
- EcoRI Digest
- Good: 6565, 3812bp
- Empty: 6303bp
Fluorescent Protein Insertion
Restriction Digests
pMSCVN-Her1
- NotI & MluI
- Digest in NEBuffer 3 + BSA at 37°C.
pMSCVN-Her3
- NotI & MluI
- Digest in NEBuffer 3 + BSA at 37°C.
CyPet
- NotI & MluI
- Digest in NEBuffer 3 + BSA at 37°C.
YPet
- NotI & MluI
- Digest in NEBuffer 3 + BSA at 37°C.
Ligation
As per NEB Quick Ligase Protocol
Transformation
- Into XL1-Blue Competent Cells
- AmpR
Validation
pMSCVN-Her1CyPet
- KpnI Digest
- Good: 4921, 3638, 2139bp
- Empty: 6346, 3638bp
pMSCVN-Her3YPet
- KpnI Digest
- Good: 5314, 3638, 2139bp
- Empty: 6739, 3638bp
___________________________________Change Log_________________________________________ PCR of the PlasmID HER1 and HER3 back bones (pDNR Dual) did not produce a product. PCR of Cypet and Ypet did
Will use Addgene EGFR WT as new HER1 backbone and Origene ErbB3 trans variant 1 as the new backbones Ordered new primers to fit these new backbone. The original H1SP specified at the top of this page is still good for use with the new Her1 from Addgene.
