Hi Nivedita, we all go through this frustration more then once in our research life, so don't feel so bad!

Ok, troubleshooting. Evidently you are not cloning, you only have background in your transformations, but low background, which is a good start at least.

So, first thing, Huseyin is right, both BspEI and XbaI are sensitive to dam methylation, check you don't have a GATC sequence overlapping one of the sites on the 5' or 3' side: the fact that your background is so low tells that you are indeed digesting your vector at least on one side. Buffer 3 is not the best for Xba, so it would be better to do sequential digestions to maximize efficiency, although I think you should have enough double digested anyway in the absence of overlapping methylation. I would not pass the vector on gel, this removes overhanging bases and reduces the capacity of re-ligation. You could use the vector as it is after heat inactivation, or just precipitate it in EtOH to clean it up a bit.

The other problem is your insert. PCR fragment, so the restriction sites are on the two ends. How many nucleotides did you leave external to the sites? To achieve best efficiency you need 3-5 external nucleotides, the RE needs to accommodate on the sequence to digest. Xba is a bastard enzyme, never digests more than 50% at DNA ends... BspEI is who knows:

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

So, it might be helpful to pass your insert into a TA vector and then digest it out from there and gel purify.

Last point... ligation of sticky ends is always most efficient O/N at 16 °C, whatever companies suggest to make you think you can speed up your experiment! It's a mix of kinetic and thermodynamic issues, but briefly, low temperature helps annealing of sticky ends, increasing the time they remain in contact, but lowers activity of the ligase, that's why longer incubation time is needed. So, if you have a difficult cloning, you'll appreciate the difference. With easy clonings, probably not..

Good luck!

Hi,

maybe this is the problem with your digest :

https://www.neb.com/faqs/1/01/01/is-xbai-affected-by-methylation

Do you see an insert that is cut out of your vector or are the sites to close to each other?

Use a non-methylating strain to prepare a new batch of your vector. Hope that helps.

Hi Nevedita,

I will assume you have antibiotic on your plates that should prevent the negative control from growing and thus any growth should be treated as either contamination of your competent cells or during your plating. Try doing a colony PCR of your gene from the test ligations to see if there may be any colonies that have been successfully transformed.

because your ligation is not ok 100%

Hello guys, My XbaI site is not affected by methylation. I ran the digested vector on gel and saw the expected fragments on the gel (apart from a blob, which I thought was undigested vector).

I was planning to do a colony PCR anyway.

Thanks

Hi Nivedita, we all go through this frustration more then once in our research life, so don't feel so bad!

Ok, troubleshooting. Evidently you are not cloning, you only have background in your transformations, but low background, which is a good start at least.

So, first thing, Huseyin is right, both BspEI and XbaI are sensitive to dam methylation, check you don't have a GATC sequence overlapping one of the sites on the 5' or 3' side: the fact that your background is so low tells that you are indeed digesting your vector at least on one side. Buffer 3 is not the best for Xba, so it would be better to do sequential digestions to maximize efficiency, although I think you should have enough double digested anyway in the absence of overlapping methylation. I would not pass the vector on gel, this removes overhanging bases and reduces the capacity of re-ligation. You could use the vector as it is after heat inactivation, or just precipitate it in EtOH to clean it up a bit.

The other problem is your insert. PCR fragment, so the restriction sites are on the two ends. How many nucleotides did you leave external to the sites? To achieve best efficiency you need 3-5 external nucleotides, the RE needs to accommodate on the sequence to digest. Xba is a bastard enzyme, never digests more than 50% at DNA ends... BspEI is who knows:

https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

So, it might be helpful to pass your insert into a TA vector and then digest it out from there and gel purify.

Last point... ligation of sticky ends is always most efficient O/N at 16 °C, whatever companies suggest to make you think you can speed up your experiment! It's a mix of kinetic and thermodynamic issues, but briefly, low temperature helps annealing of sticky ends, increasing the time they remain in contact, but lowers activity of the ligase, that's why longer incubation time is needed. So, if you have a difficult cloning, you'll appreciate the difference. With easy clonings, probably not..

Good luck!

You've received a lot of helpful answers here; I've got three small points to add:

Very good suggestions were made already! They are all worth to try or to take them into account.

Just another one for the ligation: try a "gradient": 8°C 2h, 10°C 2h up to 16°C 2 h, this can easily be done in a PCR cycler overnight!

Anyhow, although your negative control isn’t promising, I also would do a colony PCR on the 10 colonies.

Good luck, I am sure you will solve the problem!

Thank you guys. I shall let you know what happened. For your information, there is not GATC overlap on either sides. I checked it already. For my PCR insert, I have around 6-7 bases overhanging on the primer, just to make sure that the site is nesting inside. XbaI has 75% activity in buffer 3, but that works best for BspEI, this is why I added more of XbaI and left the reaction for 3 hours. i inactivated it because the last time I din't do it, and did not use AKP, I obtained 100s of colonies on all plates, clearly all background.

I was wondering if the issue is with my ligase buffer as well. nevertheless, I will try longer ligation and see if this works out. Meanwhile I shall screen the obtained colonies as well.

Thanks.

It could be the buffer - T4 DNA ligase buffer contains ATP and this does degrade with multiple freeze-thaw cycles. If you continue to get problems, then buy some more ligase and on first use, aliquot the buffer in small, single use amounts and freeze.

Ok, from what you say, it seems you have been doing things quite accurately.Very good.

I would agree always treating with phosphatase even with double digestion because you will never be sure how efficient the double digestion is, although in principle Roger's reasoning is absolutely correct... but reality can be quite different, and intramolecular ligation is hundred of times more efficient than intermolecular ligation, whatever the excess of insert.

If you wish to pass on gel your vector, fine, but keep in mind that it will partially ruin your sticky ends by hydrolyzing the single stranded overhangs, due to the basic pH of TAE and the millions of OH groups of the sugar... So you can't avoid it for the insert (but that's why you use a molar excess), be aware that you will add to inefficiency if you do it also for your vector.

Still, probably the ligation step is your real problem. I guess you had many good suggestions and you can have fun with all of them! But you point out something very important in your last post, the ligase buffer. The most delicate component of this buffer is ATP, and this is absolutely necessary for the ligation to occur. Repeated thawing and freezing hydrolyzes nucleotides, which means they lose the triphosphate group and they become useless, so it is convenient to aliquot in small volumes your ligation buffer the first time you thaw it to minimize the thawing/freezing cycles. But you can find easily the recipe for the ligation buffer on-line and make yourself a new one.

Good luck!

Haha... I guess we wrote it together... Roger definitely knows his way...

Really very useful discussion and I agree with all above Scientists. I also got frustrated when I tried cloning first time. Initially I tried cloning minimum 4 to 5 times and screened more than 500 colonies and found nothing (no expected band)  Then I used NEB Gibson cloning kit which is really good and in first reaction I successfully did cloning.I used NEB HF RE enzymes for cutting vector. Try this kit or similar and good RE enzymes (HF). Good Luck.

Ajit 

Thanks for the inputs guys, with deep dissatisfaction I regret to inform that nothing worked. I tried longer ligation, I tried longer digestions and purification as well. I even used NEB competent cells and used wonder ligation kit. Still, nothing. As a last resort, I trying my luck with sequential digestion. This XbaI is one bitch of an enzyme and I will try digestion with this one first, then increase salt concentration in my mixture and add BspEI for digestion. As control, I am going to ligate my old vector and insert, which has worked in the past and should work even this time. I hope this will really sail my boat. Oh how I hate cloning now !

This might be a silly question, but when you PCR amplify your insert, have you made sure you have extra basepairs on the 5'end of the restriction sites? I.e. say 6bp-cutting site-insert-cutting-site-6bp? If you don't have these extra basepairs, restriction enzymes will rarely work(from experience).

Cloning can be a real pain, when it's not working.

Good luck!

Yes, Mai-Brit Vadekrog. I have extra bases, but I can check again. thanks for the input

Hey, I realize that BspEI does not have sufficient bases towards the end, for it to nest on. I think this could be one potential problem.

Thanks Mai-Brit. I thought BspEI does not need this, although I did add extra bases for XbaI. I hope that this was the other issue.

Indeed Nivedita, it is not actually known if BspEI needs extra bases on the side to cut properly, as there is no mention on the NEB website, as you can see on the link on my first post... and Xba is always cutting pretty inefficiently even with 5 bases on the side... In general you might want to use higher molar ratios of your insert for ligation... maybe 5:1 or 6:1. Or just subclone in TA vector and pull it out from there...

Hi Nivedita, I'm not sure if I understood everything, but is it possible that you  dephosphorilate both your PCR product and your plasmid after digesting them? As far as I know you should only dephosphorilate the digested plasmid to allow the ligase to work properly. 

Hello everyone, I am happy to say that I could successfully clone this time. But, I would like to share my bewilderment. Firstly, the I ran the PCR insert (digested and cleaned up) and my vector (digested and cleaned) on gel to verify the concentration. I obtained about 30 ng/μL of the insert and abut 20ng/μL of the vector. I used Ligation calculator, and the amount of insert to vector for 1:3 ration suggested was about 3 ng. I realized that this was too little. Normally I do not follow this calculation. SO I ended up adding about 5μL of insert and 2 μL of vector in a 20μL reaction. The transformation yielded around 8 colonies that too on 2 different plates. Nevertheless I screened them all, and all are positive. Now, when I performed a second ligation, where I added 2 μL each of insert and vector, and transformed this, I saw more colonies. I cannot understand that if more insert is recommended, then why do I see this difference in colony numbers. Secondly, I see a silent mutation in my PCR product. The PCR mix I used has the best fidelity in the market, and yet, I see a mutation.

Anyway, I wanted to thank you all for your advices and tips, it broadened my knowledge. I also learned that less insert is not bad, especially with the proportions I was using, lesser was better.

Good luck with your work.

Nice to read that you got success in your cloning expt.