Can bacterial competent cells carry three antibiotic resistance gene on three different plasmids?

Hi everyone, I just finished my degree and now I'm trying to learn the different protocols for my Master's project. So, I apologize in advance if I am not being technical enough with my question, I'm completely new to everything but I'll try to provide everything that I know.

For the past 2 months I have been trying to clone a GOI into a specific vector that my lab uses. The vector is about ~7000 bp in length and my GOI is ~800 bp (which is contained within a different vector).

What I have been told to do by my postdoc is as follows:

For Vector;

1. Treat the vector with EcoR1 and Not1 REs at 37°C for 90 mins

2. Run it through gel electrophoresis, excise the bigger fragments and purify it

3. Treat the purified DNA with Shrimp Alkaline Phosphatase to prevent re-ligation (although I don't really understand how the vector can re-ligate if it is digested by 2 different REs)

4. Conduct an ethanol precipitation

For Insert;

1. Run a PCR with EcoR1 sequence added to the forward primer, and Not1 to the reverse primer using the vector containing my GOI. The polymerase I have tried were PrimeSTAR GXL and ExTaq.

2. Run it through gel electrophoresis, excise the GOI fragments (~800 bp) and purify it

3. Treat the purified DNA with EcoR1 and Not1 REs at 37°C for 90 mins to create a sticky end

4. Conduct an ethanol precipitation

Ligation;

1. Mix the Vector and Insert at a 1:3 and 1:10 ratio, while ensuring that the total volume doesn't exceed 5 uL. Since the Ligase I'm using is from Takara DNA Ligation Kit (Cat. 6023), I try to make sure the DNA amount matches the recommended amount (25 fmol vector : 25 - 250 fmol insert which translates to 107.8 ng vector : 12.32 - 123.2 ng insert for my particular DNA)

2. Add the same volume of the Ligase mix (total volume <10 uL), and incubate at 16°C for 3 hours - overnight

Transformation & Plating;

1. Add 20 uL of 5-alpha competent cell to the ligation mix

2. incubate on ice for >30 mins

3. Heat shock at 42°C for 30 seconds

4. Put them back on ice for 2 mins

5. Plate them on ampicillin added plates

The controls I do for the plating is;

1) Vector & Insert mix with no added ligase

2) Uncut vector of interest

3) Single-cut (EcoR1) vector of interest with no added ligase

4) Single-cut (EcoR1) vector of interest with added ligase

However, after 11 different trials, the results were always the same with little to no variations:

1:3 - no colonies

1:10 - no colonies

Control (1) - no colonies

Control (2) - A lot of colonies

Control (3) - A few colonies

Control (4) - A lot of colonies

In those 11 trials, these are the things that I have changed;

1. Used ExTaq polymerase instead of PrimeSTAR GXL.

2. Varied restriction Enzyme treatment duration from 90 mins to 30 - 180 mins.

3. Tried not doing SAP treatment.

4. Doubled, tripled, quadrupled the amount of recommended DNA for ligation reaction.

5. Incubated the ligation mix at different temperatures; 6°C and room temperature.

6. Extended the ligation mix incubation period from 3 hours to 12 and 18 hours.

7. Extended the transformation ice incubation period from 30 mins to 2 hours.

8. Added 30 uL of competent cells instead of 20 uL.

9. Successfully used TA cloning to clone the PCR product (EcoR1 - GOI - Not1) into a T vector, and then instead of using PCR products for the ligation (because we suspect that the problem might be due to low efficiency of REs on PCR products), I just treated the cloned T vector with EcoR1 and Not1 REs to get the GOI fragment in gel electrophoresis, and purified it for ligation.

I have no idea what I could be doing wrong, my post-doc only takes 2 tries of the exact same protocol to get plates with colonies (which I don't know the transformation efficiency of because I picked 5 colonies into a mini culture and all of them were false positives, and I accidentally let the plate sit in the incubator for too long so no further isolated colonies can be picked).

He said that the issue lies with my technical skills, but I did TA cloning (using Takara TA-cloning kit) completely on my own (he doesn't provide his protocols for it) and managed to get a lot colonies with 30% containing the desired insert.

He is a very busy person, so it is impossible for me to have him sit beside me and watch me do all the protocol steps. I am at my wit's end, so any feedbacks whatsoever would be greatly appreciated.

Thank you very much for reading.

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