Hello Everyone,
I am currently creating a synthetic phage display library using the Kunkel method of mutagenesis. I believe I am having problems converting my uracil containing single stranded DNA (dU-ssDNA) from the M13 bacteriophage that I am using into circular, covalently-closed double stranded DNA (CCC-dsDNA). The last part of this reaction after the degenerative nucleotide has been phosphorylated and annealed to the dU-ssDNA template is an overnight reaction at room temperature containing the annealed oligonucleotide/template mixture as well as 10 μl of 10 mM ATP, 15 μl of 100 mM DTT, 25 μl of 10 mM dNTP mix, 30 Weiss units of T4 DNA ligase (5 μl, 400 U/μl), and 30 U (3 μl, 10U/ μl) T7 DNA polymerase. The reason I believe it is a ligation problem is because after electroporating my last batch of CCC-dsDNA I observed not a single colony on my LB agar plates containing ampicillin as a selection marker indicating that the CCC-dsDNA was not supercoiled and unable to enter the cells. However, I have previously tested and confirmed that my homemade electrocompetent E. coli strain (SS320) was indeed able to survive the electroporation process and successfully take up supercoiled plasmid DNA. If anyone knows anything about how better optimize this ligation reaction it would be greatly appreciated. Thank you in advance!
-Patrick
Hi Partick,
Just thinking, what is your rationale of using DTT?
Can you substitute it with T4 DNA ligase buffer?
Hi Ms. Mwendwa,
Thank you for taking the time to help me with this. I honestly don't know why my reference protocol call for the addition of DTT. I understand that it is a strong reducing agent, but that is it. Do you believe it would be better to remove it from the reaction?
Hi Patrick,
I would give it a go with T4 DNA ligase buffer.
I hope it works.
Regards,
Leah
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