Hello Ahmet,

I think the protocol below will help you troubleshoot the problem.

You will see that:

a. XL1 will grow in LB agar plates, and that means bacteria are not a problem.

b. transformed cells electroporated with pUC will grow on both LB as well as Amp+ plates

c. transformed cells with 13kb uncut plasmid should grow on both LB and Amp+ plates

d. XL1 trasformed with your experimental (13kb plasmid that has 30nt deleted and replaced should grow on both LB and Amp+ plates)

• If no colonies seen in scenario "c" or "d" on Tet+ plates then plasmid was unable to get in XL1
• If yes colonies in scenario "c", but no colonies in Scenario "d", then you can conclude that deleting and replacing 30bp insert of your interest has rendered your plasmid with inability to grow on selection media (gene conferring Ampicillin resistance is affected), and hence no colonies on Amp+ plates

Hope the above DOE help to troubleshoot and pin the problem

Good Luck,

Savita

What do you mean with:

" I've designed primers using NEBase Changer, which don't overlap."

I do not know which cloning approach are you using but if you would like to perform deletion/insertion with a single vector PCR, priimers has to partially overlap to allow the plasmid re-closure.

I'm routinelly using the PIPE cloning approach for do the same (you can find more information about it on my blog: ProteoCool (

https://proteocool.blogspot.com/) at PAGE1 cloning or in the attached papers.

ciao

Manuele

Have you considered doing it in a two step process?

First achieving the deletion and then when successful trying to insert your new base pairs? I know it is not as convenient but I have had great success making deletions with the NEB kit. I don't think they have a protocol for doing deletions and insertions in one step?

This would require new primers and more work so you mightn't bother but if it works it could be worth it.

Savita Visal-Shah , Thank you for suggestions. I already did the control plasmid (the one that I try to mutate, 13 kb) and it is transformed and made colonies. So, competent cells and original plasmid don't have problem. Turning PCR product into replicable plasmid is problem.

Manuele Martinelli , Thanks for advice. I actually have both nonoverlapping (I've designed as suggested in NEB Q5 kit) and overlapping primers but I got the same problem. In terms of PIPE cloning, I'll look into it. Thank you. I need to figure out whether it will work for me, because my primers are in the coding sequence and I need to avoid any base insertion or deletion in order to avoid frameshift.

Niamh O'Malley I don't know whether two step approach would change the result or not. Because, in deletion step, I'll use same primers without 5` overhang (insertion). I am not sure whether this would dramatically change PCR and transformation success. I have a question though, how long do elongate? NEB recommends 1 min for 2 kb, I have followed this but couldn't get product. But haven't tested longer time, what is your experience on this?

Thank you all for helpful comments!

Efficient strategy for introducing large and multiple changes in plasmid DNA. Scientific Reports volume 8, Article number: 1714 (2018)

https://www.nature.com/articles/s41598-018-20169-8

Ahmet Alptekin Yes so I initially had some trouble optimising the reaction and ended up speaking with the NEB technical team and they advised me to use a much longer elongation time than I had tried previously. My construct is ~8kb and they suggested 4min24 which is around what you have been doing. I also found more success with just 1ng of template DNA. After these two changes I finally got PCR product (I had tried maybe 4 times with old conditions that hadn't worked). If I could see a product in the agarose gel after PCR usually the following KLD reaction and transformation was successful. If I don't see any product on the gel I don't continue on as I have never had success after that. Since then I have made about 14 different deletions to my constructs and find it a useful kit.

i have been trying the stratagene kit but no success, now i am trying the q5 kit.

can i ask your some questions?

1, is it necessary to keep the dna template amount 1ng?

2, do i need to use KLD to treat the pcr product?

3, what anealing temperature should i use?

thanks.

Hai Li

1: Recently, I contacted with NEB support about my case, they suggested me to increase DNA template ( I was using 10ng).

2: PCR product is not plasmid, I assume most of them linear and therefore, not suitable for transformation. KLD degrades template DNA and ligates linear PCR product to circular plasmid structure.

3: You can use NEBase changer to determine annealing temperature. After PCR, you can run the product on gel and see whether you have a band on right size or not. Depend on that, you can try different annealing temperatures, 1-2 different from your starting point.