User:Robwarden/Notebook/Divalent Ligand Validation/Revised Cloning Strategy

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Contents

PCR Amplification

Her1/EGFR

Primer Design

  • Sense Primer
    • 5'-gacttacg(Spacer)-ctcgag(XhoI)-atgcgaccctccgggacggc(Annealing)-3'
    • Tm = 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'
    • Tm = 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'
    • Tm = 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'
    • Tm = 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'
    • Tm = 62°C (Annealing), 87°C Overall
    • 54% GC (Annealing), 72% Overall
  • Antisense Primer
    • 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttatttgtacagttcgtccatgccgtgg(Annealing)-3'
    • Tm = 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'
    • Tm = 62°C (Annealing), 87°C Overall
    • 55% GC (Annealing), 73% Overall
  • Antisense Primer
    • 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttacttatagagctcgttcatgccctcg(Annealing)-3'
    • Tm = 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.

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