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

=PCR Amplification=

Primer Design

 * Sense Primer
 * 5'-gacttacg(Spacer)-ctcgag(XhoI)-atgcgaccctccgggacggc(Annealing)-3'
 * $$T_m$$ = 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'
 * $$T_m$$ = 63°C (Annealing), 85°C Overall
 * 42% GC (Annealing, 66% Overall

Primer Design

 * Sense Primer
 * 5'-gacttcag(Spacer)-gtcgac(SalI)-atgagggcgaacgacgctctg(Annealing)-3'
 * $$T_m$$ = 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'
 * $$T_m$$ = 63°C (Annealing), 88°C Overall
 * 58% GC (Annealing, 74% Overall

Primer Design

 * Sense Primer
 * 5'-GGCGGAGGGGGTACGCGTGGGGGCGGCGGTAGCGGCGGGGGAGGTTCC(Linker w/ MluI)-atggtgagcaagggagaggaactg(Annealing)-3'
 * $$T_m$$ = 62°C (Annealing), 87°C Overall
 * 54% GC (Annealing), 72% Overall
 * Antisense Primer
 * 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttatttgtacagttcgtccatgccgtgg(Annealing)-3'
 * $$T_m$$ = 63°C (Annealing), 75°C Overall
 * 46% GC (Annealing, 57% Overall

Primer Design

 * Sense Primer
 * 5'-GGCGGAGGGGGTACGCGTGGGGGCGGCGGTAGCGGCGGGGGAGGTTCC(Linker w/ MluI)-atggtgagcaaaggcgaagagc(Annealing)-3'
 * $$T_m$$ = 62°C (Annealing), 87°C Overall
 * 55% GC (Annealing), 73% Overall
 * Antisense Primer
 * 5'-gacttcag(Spacer)-gcggccgc(NotI)-ttacttatagagctcgttcatgccctcg(Annealing)-3'
 * $$T_m$$ = 62°C (Annealing), 74°C Overall
 * 46% GC (Annealing, 57% Overall

Reaction Cycle
=ErbB Insertion=

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

pMSCVN-Her1

 * EcoRI Digest
 * Good: 6581, 1838, 797, 768bp
 * Empty: 6303bp

pMSCVN-Her3

 * EcoRI Digest
 * Good: 6565, 3812bp
 * Empty: 6303bp

=Fluorescent Protein Insertion=

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

pMSCVN-Her1CyPet

 * KpnI Digest
 * Good: 4921, 3638, 2139bp
 * Empty: 6346, 3638bp

pMSCVN-Her3YPet
___________________________________Change Log_________________________________________ PCR of the PlasmID HER1 and HER3 back bones (pDNR Dual) did not produce a product. PCR of Cypet and Ypet did
 * KpnI Digest
 * Good: 5314, 3638, 2139bp
 * Empty: 6739, 3638bp

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.