I am trying to replace a 2.6Kb fragment from a 8.9 kb vector with a 2.7Kb insert. I am digesting my vector with EcoRI and SacII, and clearly seeing a fall out of 2.6 Kb and remaining 6.3 Kb of vector backbone. So, I am sure that my digestion is at least >90%. My insert is generated through golden gate assembly followed by PCR with primers having EcoRI and SacII site.
I am preparing a master mix of Digestion buffer and enzyme first and then using the same master mix to digest both my vector and insert. I am doing SAP treatment for my double digested vector too. My insert also have 3 extra bps between terminus and restriction site for efficient digestion. But after ligation, I am hardly finding any colonies (Efficiency of my comp cell is around 10^6 CFUs). When I am screening those 3-4 colonies I am getting after transformation, they are not coming positive. I have repeated this experiment only god knows how many times with sequential digestion and different ligation parameters. But nothing seems to work. Can anyone please tell me what might be going wrong?
Thank you so much in advance.
Hello,
the general rule of thumb I was taugth is to let A 6 nucleotides overhang at the end of your insert before the restriction site to ensure that the enzyme cut properly.
Here is the NEB chart for each enzyme:
https://www.neb.com/en/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
Maybe try to double or quadruple your digeston time of the insert to be sure.
Do you check your insert on a gel after the PCR adding your restriction sites ?
Lastly, as I have myself had a lot of problem with goldengate, I know how you can become pretty desperate haha !
One thing that worked for me was to let my transformed bacteria grow at least 48h to be sure of colony presence. I don't know the reason.
Best of luck,
Philippe.
Hi Philippe Paget-Bailly
Thank you for your suggestion. Yes I have checked my insert in gel after restriction site adding PCR. I saw clear amplification. And after gel elution of that PCR product also I had the desired size band. This time I have done 20hr digestion of insert with SacII specifically.
Keeping your suggestion in mind, I am letting my plates to be in 37C for at least 36hrs. Hoping to see some positive colonies this time.
Thank you.
Do you check your insert on a gel after the PCR adding your restriction sites ?
Hello @Nahla Hammok,
Yes I did. My restriction sites are definitely added, otherwise I wouldn't have seen the amplification. Although my template and amplicon size is almost same, I took less than 5ng of template per 50ul reaction. There was no way I could see DNA, If not amplified product.
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