Now I need anyone's help, regarding linker ligation to vector, whichI tried several times in vain.
In order to create EcoRI site just before NcoI site of Promega pGL4 vector,
I ordered two combinations of linker oligos as follows.
EcoRI-SalI-NcoI linker sense: 5'-catgcGAATTCGTCGAC-3'
EcoRI-SalI-NcoI linker antisense: 5'-catgGTCGACGAATTCg-3'
EcoRI-BamHI-SalI-NcoI linker sense: 5'-catgcGAATTCGGATCCGTCGAC-3'
EcoRI-BamHI-SalI-NcoI linker antisense: 5'-catgGTCGACGGATCCGAATTCg-3'
BamHI and SalI sequences are not essential, but I included them considering possible enhancement of hybridization between sense and antisense oligos.
I made the 100 uM TE solution of the oligos.
To make sense and antisense oligos hybrize to each other, I mixed the same volume (10 ul each) and heated them to 99 ? for 5 min, then left them to room temperature for a few hours.
I linearized the pGL4 vector by NcoI, CIAP-treated and comfirmed the band by gel electrophoresis.
Ligation was performed using 100 ng (?0.03 pmol) of pGL/NcoI+CIAP and 50 ng hybridized oligos (?5 pmol), using NEB T4 ligase.
As a control, I prepared reaction without oligos.
Colony number is almost identical and low (around 100 per plate) regardless of the presence or absence of oligos.
I checked dozens of colonies but none had EcoRI site.
How can I solve the problem?
WIthout this step successful, I cannot proceed to the next step. Please give me advices.
Thank you in advance.