1. You can solve your issue with the insert and plasmid being the same size by finding an enzyme that cut inside the plasmid but not the insert so the plasmid fragment will be broken to several pieces.

2. If you just cut the ends of a PCR product and linearizing a plasmid you can use PCR purification instead of gel extraction since the yield is usually better.

3. XhoI require at least 4 bases before the site to work efficiently, if you don't have that you can just add extra bases to your primer before the restriction site.

4. For phosphatase treatment, I just add it to the restriction reaction and let it sit for an additional hour or so, I don't bother with the inactivation since most of it will be gone in the purification step.

Are you getting no colonies after transformation or a bunch of colonies but none are correct?

If the first then you should be sure that your competent cells are good. I would expect that you have a background of pUC57 in the transformation since there is always going to be a little bit of it as the PCR template.

Have you tried transforming a with a control plasmid? That would rule out issues with the competent cells.

Also, you are probably losing most of your product with all of those cleaning steps. Did you try running out any of the plasmid + insert on a gel after the ligation step?

Use a heat-inactivated enzyme and phosphatase. Don't bother to do a gel purification. You don't need to do a column clean up after every step either.

1. Measure concentration of your plasmid, then do a digest combined with phosphatase, then heat inactivate.

2. Set up your PCR, run tiny aliquot on gel to estimate concentration/make sure you have just 1 band.

3. Do math to see what volume of plasmid + PCR you'll use. 1:1 ratio is good since they are same size.

4. Digest PCR, then heat inactivate.

5. Set up ligation. Modern enzymes will work in 5 minutes, but overnight is fine. Check the kit for the appropriate temperature.

6. Transform cells.

My guess is that you are seeing no colonies because you have lost too much of your starting molecules.

Good luck!

as Yoav Lubelsky wrote you can avoid to PCR the fragment if you cleave the plasmid with an enzyme that is not in your insert (and even better in the ampicilline gene...)

also if your destination plasmid gives kana resitstance (or any other exept ampicillin) you just plate your ligation on kana (E. Coli with puc57 will not grow)

for dephosphorylation use an antarctic phosphatase that is easely inactivated (don't need to purify the vector)

also did you have a look at Gibson's cloning? (see NEwBuilder on NEB site.. very efficient and less work ) (there is probably other kits as efficient but I use that one)

Yoav Lubelsky

Thanks for sharing your experiences.

As Michael implies, due to the presence of tiny pUC57 as the PCR template, I had to purify the PCR product for prevention of pUC57 intervention and false colony background. On the other hand, gel purification led to reduced DNA yields. Additionally, I tried a cleaned-up PCR product and got a lot of colonies with pUC57.

XhoI restriction sites in designed primers are optimal, and I have tried different Alkaline phosphatase treatment times at 10, 15, 20, and 45 min to make sure dephosphorylation takes place and then gel purification (or 65C Enzyme deactivation). I got a few colonies that had not desired insert. I even transformed these cut and dephosphorylated plasmids alone, and I got a handful of colonies. So, Alkaline phosphatase works well.

According to your advice, I’II try digestion, especially in Plasmid AmpR sequence to digest remained pUC57 in PCR product. Is it okay to try it before PCR amplification or after that? And does it need gel purification or clean up before cloning RE treatments or not? or I digest pUC57 simultaneously with the insert?

Thenks for your response Michael J. Benedik

My competent cells are efficient according to control plasmid transformation. Your expectation is true, and I experienced a lot of colonies with pUC57 when once I tried clean up instead of gel purification in the insert preparation step.

Thanks for your response Katie A S Burnette

My competent cells are qualified. Due to the variation of plasmid size in electrophoresis, I thought it may have confused me so I hadn’t tried it yet. Is it useful?

Your guess is true about low DNA yield due to multiple purification steps, and I try to reduce it by heat inactivation instead of gel purification.

Thanks for your advice, Didier Poncet

Unfortunately, I can’t use Gibson assembly due to the limited overlapped region between my synthetic sequence and plasmid. According your advice, I’ll try digestion plasmid with pUC57 cutter enzymes.

Thank you.

Based on my previous unsuccessful closings I want to prioritize double digestion cloning via primer changing. Do you have any advice about cloning and ligation with Xho1, Nde1, or Kpn1?

In your opinion, for DNA yield preservation what enzyme inactivation method is better to use between my treatments (Xho1, Kpn1/Nco1, and pUC57 cutter enzyme treatments)? Heat inactivation, gel purification, or clean up?

If I find a compatible and common buffer for two enzymes, will simultaneous treatment interfere with RE operation?

I'd suggest you use the NEB online tools (assuming that's where you buy your enzymes).

Here's the advice page: https://www.neb.com/en-us/tools-and-resources/usage-guidelines/double-digests

And the Double Digest online tool: https://nebcloner.neb.com/#!/redigest

for dephosphorylation we use the rSAP (from NEB but maybe there is other brand); it is a shrimp enzymes works at 37°C in the restriction buffer and is easy to inactivate. With other phosphatases (like CIP) you can get zero colonies background because the ends of the vectors are damaged by the phosphatase and the phosphatase sticks on the ends of the DNA and is not easyly removed or inactivated... (and you get zero colonies with the ligation)

for the gibson assembly you only have to redesign new (longer) oligos ; once you tried it you won't go back to restriction/dephosphorylation/gel purification/ligation/transformation/incubation .... disappointment !

yes go ahead

Thanks to all of you.