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 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.
With vector step 3, that is to prevent blunt end ligation which occurs with the overhang undergoing degradation.
With the insert step 1, do you have some bps on your primers before the added restriction site?
Your transformation protocol is questionable. Usually you add 1-2µl of ligation mix to the competent cells. After step 4 you need to incubate the cells in rich media at 37°C for 1h to allow them to recover and express the genes for antibiotic resistance before plating.
Overall it seems the problem is with your ligation step or your GOI is toxic when driven by the promoter in your destination plasmid. You tried adding PEG to your ligation mix? Another trick is to combine your vector and insert fragments and then use a PCR machine to warm to 95°C for 10 mins before putting on ice. This is to remove any secondary structure that might interfere with the ligation.
With the ethanol precipitations how long are you drying the pellet for? If you dry for too long you can cause degradation of the fragments.
Do a PCR on the ligation mix across the vector MCS to see if you are forming your desired product.
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