Make sure the product is not degraded, fresh amplify, make sure sufficient amount of insert (do not go with the OD, always gel run) is present (make 50 ul PCR reaction volume if required)

Use all your products in the ligation reaction and corresponds your vector concentration, Ligation time should not be the problem.

After transformation, incubate the cells in 1 ml LBB, centrifuge, then resuspend in around 20 ul LBB, and plate.

and plz use freshly prepared antibiotics

See if it works

Dear Harshita Tiwari

If i undertand well. you amplified the insert adding the restriction site regions and did you performed a standard ligase? Is it correct?

Which site did you used?

However as general comment, to insert so small region you can avoid to amplify and clone an insert but you can just perform the plasmid PCR using a stratregy similar to single point mutagenesis. You have to add in the primers a region with codify for the region to be inserted (with overhangs) using the PIPE cloning strategy

you can find an example of it at the following link:

https://proteocool.blogspot.com/2021/09/tips-of-week12-rapid.html

good luck

Manuele

if I understand you try to clone 65 long oligos?

first: double check the sequence you synthesized !! then

it is important to rehybridized the two oligos (after boiling the mix and slowly return to ambiante temperature) and the proportion of vector/insert should be low (if the oligo are not phosphorylated then an exces of oligo will add oligos on both vector ends and they could not close the circle (because they are not phosphorylated)

is it with two different restriction sites or only one? I would prefer with 2 restriction sites to get rid off the wt vector background

are the restriction site(s) reconstitued after ligation with the insert? if not then you can digest the ligation product with this restriction enzyme to reduce the background

with the dephosphorylation of the vector+phosphorylation of the insert you can get clones multiple oligos inserted.

One of your restriction enzymes might not be playing ball. A positive control should tell you if that's the case. I generally prefer to digest the insert rather than go down the phosphorylation route - fewer things that can go wrong, in my experience. But if everything's alright, your strategy should be fine.

Kindly give importance for the sequence synthesized

If you’re not getting colonies after transforming your plasmid with a ligated insert, several factors could be causing the issue. Here are some common reasons and troubleshooting tips:

Possible Reasons & Solutions:

1. Inefficient Ligation

• Insert-to-vector ratio: Ensure an optimal molar ratio (typically 3:1 insert-to-vector).

• Ligation conditions: Use a fresh T4 DNA ligase and incubate at the correct temperature (e.g., 16°C overnight or 1 hour at room temperature).

• Vector dephosphorylation: If the vector was dephosphorylated, ensure it was necessary; excessive dephosphorylation can prevent ligation.

2. Poor Transformation Efficiency

• Competent cells: Use high-efficiency chemically competent cells (>10⁸ CFU/µg DNA) or electroporation for better results.

• DNA quality: Ensure clean, properly ligated plasmid DNA.

• Heat shock/electroporation conditions: Optimize the transformation protocol (e.g., heat shock at 42°C for 45–60 sec, proper recovery time).

3. Vector Re-ligation (Self-Ligation)

• If the vector re-circularized without the insert, it may lead to no or very few colonies.

• Control: Run a negative control with only the cut vector to check background colony formation.

• Solution: Use a proper insert-to-vector ratio and check if the insert was phosphorylated (if necessary).

4. Insert Integrity and Compatibility

• Restriction sites: Confirm the correct digestion using gel electrophoresis.

• Insert sequence: Check if the insert has internal restriction sites preventing correct ligation.

• Compatible ends: Ensure cohesive or blunt ends match the vector’s ends properly.

5. Antibiotic Selection Issue

• Incorrect antibiotic: Make sure you are using the correct antibiotic and concentration.

• Non-functional resistance gene: If the plasmid backbone has been disrupted, resistance may be lost.

6. Toxic Insert

• If the insert is toxic to the host cells, colonies may not grow.

• Solution: Use a lower-copy plasmid or inducible promoter system.

7. Incorrect Plating Conditions

• Incubation time: Some plasmids require longer incubation times (e.g., 18–24 hours).

• Incorrect media: Ensure you are using LB with the correct antibiotic and supplements if necessary.

Suggested Controls to Diagnose the Issue:

1. Vector only (no ligase): Checks background self-ligation.

2. Vector only (with ligase): Ensures ligation works.

3. Insert only: Should not produce colonies.

4. Uncut plasmid transformation: Checks cell competency.

What molar ratio did you use in your reaction, and how many total nanograms of DNA did you use?

With such a small insert going into such a large vector, it might not be a bad idea to try upping your insert-to-plasmid molar ratio to something closer to 5:1 or even 8:1, which should increase the chances of an insert finding a vector.

Depending on how many total nanograms of DNA you added to the reaction, you might consider upping that as well.

Also, to make your life easier, you might find it useful to use this ligation calculator tool to help you set up your recipes: https://dnatutorfiles.shinyapps.io/LaneCalculator/

Good luck!