Hi Manshi,

I cannot answer your question directly - it seems a little bit odd that you get colonies from your ligase absent and no or little colonies from your ligase present control when you use the same amount and batch of your digested vector, because if you have undigested vector present you should see roughly the same amout of colonies on both plates. However, as you are working with small amounts of material some variation might occur to due to pipetting inaccuracies.

Nevertheless, I think the more important question for you is why you don't get positive clone from your ligation with insert + vector.

Can you give some more information what you are doing?

What restriction sites do you use?

Are you trying to clone a PCR product or do you cut out your insert from a different vector?

Which vector do you use?

How is your molar ratio between insert and vector?

How do you analyse your clones after ligation (colony PCR or digestion)?

With this information maybe I can get an idea what might be the reason you don't get positive clones (however I cannot guarantee this).

KR Dörthe

Hi KR Dörthe,

Thank you so much for responding. Yes, I can provide you the details of what I have been doing for past 6 months. I'll answer each one of your question.

Answer 1: I am actually trying to insert a PCR product of 2.3kb size with restriction sites at the ends in the vector called pAd-Track-CMV which is of 9.2kb size.

Answer 2: Restriction sites that I am using are SalI and XbaI. In the insert, I introduced these sites during the primer designing process. SalI in the Forward Primer and XbaI in the Reverse Primer. I've also added extra sequences at the 5'end for the restriction enzymes to work properly. And for the vector, it's present in the MCS.

Answer 3: I am trying to clone a PCR product.

Answer 4: The vector is called pAd-Track-CMV. It is of 9.2kb.

Answer 5: This is going to be interesting. Earlier I used to perform ligations in 3:1, 5:1 ratios. But as I mentioned this to my PI, he ordered me to not go with the ratios. Just 1ul of vector, 1ul of ligase (as per the NEB), 10ul of ligation buffer (as per the NEB) and whatever volume is left from 20ul reaction add the insert completely (no place for AMQ). His concept behind this was that by adding the AMQ you are diluting it. So after that I started adding complete insert in it. Now my reaction is Vector=1ul, Insert=8ul, ligase=1ul, and buffer=10ul. Incubation for 15 minutes and then transformation. Recently I got around 94 colonies in my vector + insert plate but no clone yet.

Answer 6: I screen my colonies through Restriction Digestion with the same enzymes. I inoculate the colonies, grow primary culture, isolate the plasmid and proceed for double digestion. Then I run the gel.

PS: I've also added few images for you reference.

I'd be very grateful if you can find out the problem in it. Thank you so much for your precious time.

Manshi

One possible reason for not getting colonies in your vector-only (ligase present) plate could be inefficient re-ligation due to dephosphorylation or other ligation-inhibiting conditions. If you’re using calf intestinal phosphatase (CIP) or similar enzymes to treat your vector, ensure it's not over-treated, as excessive dephosphorylation can hinder even minimal self-ligation, which may explain the absence of colonies in this control. To improve ligation efficiency in your insert+vector reaction, consider adding 5% PEG 8000 (from a 25% stock) to your ligation mix—this enhances molecular crowding, thereby increasing the chances of vector and insert coming together. It’s also advisable to optimize the vector-to-insert ratio; using the NEB Cloner tool can help determine appropriate molar ratios. Try both 3:1 and 5:1 insert-to-vector ratios, as sometimes small changes in stoichiometry can make a big difference, especially with larger vectors like your 9.2 kb construct.

Hi Manshi,

sorry that it took me sometimes before I come back to you. I think your cloning strategy looks ok, however I have some recommendations.

The first think I want to ask you if you tried to digest your DNA from your colonies with other restriction enzymes (eg EcoRV and KpnI which are flanking your restriction site) than the enzymes you use for the ligation? Or have you tried colony PCR?

I am not sure if you are aware that the Xba I is prone to overlapping dam-methylation. If the restriction site is methylated the enzyme will not cut. If you are unlucky you might have created a methylation site due tothe flanking sequence of your primer - you should check for this (you can look this up here https://www.neb.com/en/faqs/0001/01/01/is-xbai-affected-by-methylation?srsltid=AfmBOorYpT5WcWZt2dCkB--z0brSq5x3fPRfEyIzSHG-vh3GEJ5-ztue) - check this first before trying anything else as this is easy to check.

If this is not your problem there are several things you can try. I usually I did a sequential restriction with only one enzyme at a time to see that both enzymes are really cutting - in between I did precipitate the DNA with Lithium acetate and ethanol ( I always did 2 restriction in your case one with XbaI first and SalI second and vice versa). If possible limit the amount of time your DNA is exposed to UV-Light. I would dephosphorylated the vector after you digested the vector with both enzymes, especially as with this approach you will still have the small fragment you cut out from the vector in your reaction, which can promote religation if not dephosphorylated.

As an alternative you can run the digested vector on gel and purify the vector from the gel to get rid of the small vector fragment - in this case it might not be necessary to do a dephosphorylation as the XbaI and SalI site should not religate. However, if you cut out the vector from the gel minimize the time your vector is exposed to UV-light as prolonged UV exposure might damage the DNA and decrease transformation efficiency.

I would at least use 50 ng of vector for ligation as your vector is relativly big - I would also reconsider using the molar ratio as I think this is also recommended by NEB (in your case this would mean 37,5 ng insert and 50 ng vector for a 3:1 molar ratio)

As Debajit Das recommended you can also try to add PEG - however I am not sure if this will work with the quick ligation kit.

You might also try to subclone your PCR product in another vector first like TOPO or pJET which are designed for direct cloning of PCR products - sometimes it is easier to clone an insert you cut out of another vector than cloning the PCR product directly (and in this case you can sequence your insert first before cloning it in your target vector) - if possible I would recommend to try this approach maybe in parallel to your approach to clone the PCR product directly.

You can also try another ligase (not the quick ligase) and ligate the insert at lower temperature for a longer time (eg at 16°C over night or at 4°C for 48 h).

Another thing is that there is always the possibility that your insert is toxic for E.coli and you don't get positive colonies for this reason. In this case you can try to grow the colonies at lower temperature or use another strain of competent cells.

I know this is a lot - if you have additional question feel free to ask.

KR Dörthe

Hi KR Dörthe

Thank you for responding.

Thank you for the XbaI information. For my further transformations I would use different E coli strain as mine is dam+.

And for today's screening I'll use the restriction site flanking my SalI and XbaI sites as I've already transformed my ligation reaction into dam+ strain.

For the sequential restriction digestion, I think that is not the problem as both of my enzymes are fresh, working properly (checked using SalI + PmeI separately and XbaI + PmeI separately. PmeI is present in my vector) and also both of my enzymes are compatible in r3.1 buffer. And after digestion I usually purify both of my vector and insert using the purification kit and then proceed for ligation.

For the ratio part, I think I should be doing my ligations in either 3:1 ratio or 5:1 ratio parallelly.

I cannot use PEG as it is already present in my ligation buffer and exceeding its concentration would severely hinder my transformation.

I cannot subclone my PCR product in another vectors as this vector will be further used for Homologous recombination with pAd-Easy system (this will generate adenoviral backbone which I would be using in mammalian cells) so cannot change the vector. And for the insert part, I cannot clone my insert after cutting out of other vector as I have added HA tag after my sequence (added while designing the primer).

But definitely I would change the E coli strain.

Thank You so much for your valuable suggestions. I'll definitely keep these information in mind. And I hope if I find any difficulty doing it, I can reach out to you.

Manshi

Hi Manshi,

just one additional question - when you purifing your vector are you sure you get rid of the small fragment you create when cutting your vector with the two enzymes (this is not clear for me from your answer)?

This would be important, because if this is still in the reaction it will perferably ligate with your vector (instead of your big insert, especially when you not dephosphorylate your vector) and in the end you will get the original vector.

If you are not doing it already run the vector on a gel and cut the vector band out to get rid of this small fragment - however avoid prolonged UV exposure. The other possibilty is to dephosphorylated your reaction after digestion - this will also dephosphorylate the small fragment and avoid religation of this fragment in your vector.

You can reach out to me at any time.

KR Dörthe

Hi KR Dörthe

After purification of my vector I never thought about the little fragment. Infact it is so small that it cannot be seen in 1% gel so never occurred to mind.

However yesterday, I digested my isolated plasmids with SalI and XbaI along with that I also digested those plasmids with KpnI + EcoRV. I observed a fall out in one of the screened plasmids. In the SalI + XbaI double digestion screening there was a faint band at 2.3kb but in the KpnI + EcoRV double digestion screening the band was quite dark but near 3 kb. How's that possible when KpnI and EcoRV sites are so near to SalI and XbaI sites respectively? Also I've added HA tag sequence but that would also not affect the size this much.

And also how can I prove it with the SalI and XbaI because they are producing faint band?

I can show you the image for your reference.