1. Neon Revival Through
Gibson Assembly
SAMENDER RANDHAWA, JOHN ZHANG AND DR. DANIEL P DOWLING
SUMMER OF 2015 DOWLING LAB
2. Outline
Introduction To Gibson Assembly
Primer Modification
Reactions With New Primers
HF Polymerase vs Taq Polymerase
The Temperature Variable
Nuking The PCR
Optimizations for PCR
Estimating DNA Content
Present Day Attempts
Future Steps
3. Gibson assembly
Joins multiple DNA fragments together in a single
reaction
Components:
5’ exonuclease
DNA ligase
DNA polymerase
NEB Gibson Assembly:
No clean up steps required
DNA can directly used for transformation in to E. coli
cells (NEB5 α cells)
Tobias Vornholt
6. Future Steps To Obtain NeoN
Colony PCR with a few isolated colonies and then run an analytical
gel to verify the presence of NeoN gene
Taq polymerase was used to check for the size of the insert. We are
not concerned if all the bases are present or if mutations occurred.
Cheaper way to check for size of the inserts. Also the T7 promoter
and T7 terminater primers were used as these corresponding regions
are present in the pET28a vector
Prepare the colonies to be sequenced
Analyze the sequence of the DNA sequences
