Hi All,
Recently I generate a vector (~6k bp, amp resistence) which only have EcoR1 and EcoR5 in MCS and can let me insert cDNA between this 2 enzyme. and This vector has been sent to sequencing, and confirm it has this 2 enzyme cutting site. So I do subclone and try to put a insert cDNA (~1k) between this 2 site, I got many colonies E.Coli (stbl3) on my LBamp plate, and feel very happy, But after I pick the colonies, mini-prep and PCR, the PCR results shown no insert, but only vector.
the whole step is like this: PCR insert which has EcoR1 and EcoR5 site. also my vector has EcoR1 and EcoR5 site. then I cut both of them(2ug each) with 2ul EcoR1 and 1ul EcoR5 overnight at 37C with NEB buffer 3.1. total 50ul. Next day I run the gel, cut the correct size band and purify it. The bands I get was in correct size and the CONC was enough. Then I set up ligation with rate insert/vector 3/1 with T4 ligase, RT 1h. Then I heatshock the ligation product into stbl3 and shake it in SOC, sketch on LBamp plate and get many colonies (~20/plate) next day.
First I though maybe the RE cut was not fully, so that I get the vector uncutted and heatshock it into my stbl3, So I tested the Ecor1 and Ecor5 with same buffer, but just 1 h 37C, separately with another enzyme also in my vector, and results shown it fully cutted in just 1 h, as I did subclone RE cut 37C overnight, I would like to think it's fully digested.
But As Ecor1 and Ecor5 was different enzyme, it shouldn't link each other after cut, but my subclone reuslts shown only vector. and I did this subclone 4 times, also with a different insert, but get same results, really need help!
Thanks
Monica
Hi there,
The main issue is that you get religation only which means you have a problem with the double digest efficiency: self ligation occurs with plasmid digested with a unique RE.
Here are a few lines of thinking:
How far away are the two restriction sites in the vector? If too close both enzymes may interfere. Sequential digest might help.
Have you run the ligation controls (plasmid alone without or with ligase in the ligation mix) to evaluate non digested plasmid and single digested plasmid in your prep? Furthermore comparison with full ligation mix would tell you if the insert has any effect on the number of clones (measuring the ability of the inser to interfere with self ligation).
Are you sure about digest efficiency on your PCR product? Are the sites very close to DNA ends? It mayy affect cleavage efficiency (https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments).
Hello Monica,
I don't know wich vector are you using, but as I can understand you have an empty (no insert at all) vector, and you try to put a PCR product in there, right?
The problems I see with your aproach are, first, some restriction enzimes need some space after and before the recognition site, so they can properly bind to the molecule, so in the case you are trying to digest the PCR product that has restriction sites in the borders, maybe it's not enough space for the proper digestion. This can be esealy solved using a cloning vector like pGem-T, and all his vaariants, since the only thing it needs to ligate to the PCR product is the presence of Adenine nucleotides (added by the polymerase if it's a regular Taq or TaqPlatinum, not Pfu) and then you can digest the insert from this vector and ligate it into the one you actually need.
About the vector, if it's religating it's obvious that both enzimes are not working, if you are using an empty vector you will not be able to see it, since the only thing you can know is it's linearizing, but not if both enzimes are working. You could try to do an essay with each enzime in diferent reactions to check that both are properly working, and also checking that the restriction sites are not too close to each other, if it's the case, again, you may have problems.
One easy thing you could do is not using an empty vector, but one that already has an insert on it, so you can check digestion is going well by seeing the insert being relased after cutting with the enzimes, cutting the band with the expected length of the empty vector, and go as you know.
Hope it helps,
Edward.
1. cut your vector sequentially with RE. don't gel purify. do pcr purification.
2. increase the insert conc and use neb cloning tool for concentration calculation. Preferably 5:1.
3. if you are still getting self ligation. do forced cloning.
Hi All,
Thanks for many advise, I have done the ligation controls yesterday, the only vector RE product heatshock yield no colonies, and the only vector, RE cut and ligation yields many colonies, it seems my vector ligation itself after double digest. As sequencing results shown there is only one RE site for each enzyme, I feel the reason may due to 2 enzyme too close. I'll try a Sequential digest next week to see if better result.
Many thanks!
Monica
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