Recently trying ligase based cloning using T4 ligation. I digested the donor plasmid and linearize the recipient with the same enzyme (double digestion) to generate cohesive ends. The insert was gel purified for ligation reaction. The reaction mix was prepared in insert: vector molar ratio of 2:1. I used 100ng of vector for 10ul of the final volume. Ligation was made at room temp for 5 min. Then half volume used for transformation. Transformation succeeded with an insignificant background. To confirm ligation of GOI, plasmid extracted from transformants and double digested with the same enzymes. However, I couldn't get band on gel electrophoresis. How could I finely tune? I wonder if anyone suggests improving my experiment.
Try to first confirm the insert using PCR (pref 1 primer in backbone and 1 primer in insert) this rules out mutation of restriction site post ligation. If this turns out to be the case then simply more colonies and run the digest again.
Certain inserts are not stable and will cause plasmid to be rearrange (digest the vector and the minipreped plasmid using different restriction enyzme than the ones used to clone in order to confirm the plasmids you are getting are truly just empty vectors). If this turns out to be the case change the background strain and do the recovery and overnight playing at room temp or 30 (try neb stable, neb10, stbl3 as examples)
upload the names of the enzymes you used for the digest you could be using two different enzymes but they could have compatible overhangs which means your vector will simply religate, however, this will destroy the sites of both enyzmes thus you will not even get a linear fragment after digestion.
check the distance between the restriction sites in case they are very close you might need to use different sites as the vector might be getting linearized by a single enzyme.
Other problems could be related to the selection antibiotic (especially if you using ampicillin) make fresh plates and fresh antibiotic stock.
Best of luck!
You are missing a critical step in your cloning procedure : vector dephosphorylation.
Since you digest your vector with only 1 enzyme (even if it cuts at 2 sites), your vector may reclose itself without incorporating the insert when adding ligase.
To avoid so, treat your vector with Alkaline Phosphatase or CIP phosphatase.
Dephosphorylation removes a phosphate from the extremities of your vector, which makes it impossible to close itself even with T4 DNA ligase. It does not affect ligation to an insert however.
For transformants screening, you can quickly perform "colony PCR" against your GOI instead of growing and extracting DNA from each clone. For that, resuspend a small amount of each colony to test in PBS, and use 2ul as a DNA template for a regular PCR. Increase the initial denaturation step to 3-5mins to allow lysis and release of DNA.
Dear Martin,
Thank you for your suggestion. Regarding phosphorylation, I cut the vector and insert with two different enzymes, EcoRI & XbaI which recognizes different sequences so dephosphorylation could not be a problem. Again I sceptic of the colony PCR, because I already succeeded construction of expression vector. This case only preparing the MOCK. Again thank you for your suggestion. I will consider insert specific amplification by colony pcr.
Sintayehu Fekadu Sorry I misunderstood your problem.
So, you don't see the expected band when you double digest your transformed clones. Depending on the size of your GOI, you may see a shift in total plasmid size with a single cutting enzyme (to have the linearized size). If the total size is concordant with the insertion of your GOI, then the problem may be your enzyme stocks.
As for your cloning procedure, everything looks fine. I personnally go for a 3:1 ratio and perform the ligation step for a minimum of 30mins at room temperature (or at 16C overnight), but 5 minutes should work just fine considering you are not doing blunt-end ligation.
Are you using an antibiotic resistance gene to select for the insertion of your GOI ? What do you mean by "insignificant background" ? Have you tried doing a control ligation without the insert (only vector + ligase) ?
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