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Nde1-BamH1 makes me sad


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34 replies to this topic

#16 jiajia1987

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Posted 10 March 2010 - 07:28 PM

Sorry, NdeI not NheI. If you cut a linear fragment (PCR product, e.g.) with NdeI only, then one end is cohesive to other NdeI sites, the other end is inactive. If you ligate, you get double length fragments, if the cutting and religation works. Same with the BamHI site at the other end. No other side reactions occur, so you can easily determine the cutting and religation efficiency.


Cool, I get it now! Thanks!! :P :lol:

#17 Denny

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Posted 10 March 2010 - 07:48 PM

Sorry, NdeI not NheI. If you cut a linear fragment (PCR product, e.g.) with NdeI only, then one end is cohesive to other NdeI sites, the other end is inactive. If you ligate, you get double length fragments, if the cutting and religation works. Same with the BamHI site at the other end. No other side reactions occur, so you can easily determine the cutting and religation efficiency.



Very slick phage!!! What a great time saver!


"Thanks for the recipe reference! Just a question here, you stated 10ul of DNA, how much of the DNA would that be? How many ng or ug?"

I'm not real critical on this point, unless I'm having problems. In which case, I just check the NEB recommendations.
I get anywhere from 60 ng/ul to 250 ng/ul from mini-preps of vectors and use them undiluted. If your concentration is high, definitely check the recommendations and dilute as needed.

My favorite combo double digest team is XhoI and NdeI. The play very well together. But we must work with what we have. So onward and upward with BamHI

Now that you've got brain burn, I want to clarify something real quick in case it's not obvious with everything we've thrown at you. My troubleshooting ideas are directed at cutting the original pet-22b vector NOT the new linear PCR version.

And after reading this post:
http://www.protocol-...osts/18976.html

My autopilot turned off and I realized that you can skip the CIP step after the second digest.

Phage has some awesome time saving suggestions! I'm taking notes! :Dbiggrin.gif

Dukey is absolutely correct in that some combinations will just give you fits. They don't all work and they don't all work easily. Consider another vector with Dukey's information about restriction sites if you can't solve this
in a couple weeks tops!

Edited by Denny, 10 March 2010 - 08:42 PM.


#18 jiajia1987

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Posted 10 March 2010 - 09:36 PM

Thanks to everyone for all the awesome ideas!

And one more thing before I start on all the ideas here! --> What temperature do you guys do your ligation at? 16degC? I always it at that temp overnight.

#19 Denny

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Posted 11 March 2010 - 04:38 AM

Thanks to everyone for all the awesome ideas!

And one more thing before I start on all the ideas here! --> What temperature do you guys do your ligation at? 16degC? I always it at that temp overnight.


I've always had good luck with Quick Ligase for 5-10 min @ room temp
Here's the particulars from NEB on T4 which I don't remember using in a very long time.

http://www.promega.c...180/9pim180.pdf

The methods and protocols I use allow me to run pcr, digest, ligate, transform in the same 8hr. day.

#20 phage434

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Posted 11 March 2010 - 06:09 AM

I use normal (not quick) ligase for 15 minutes to 30 minutes at room temperature. For some cut sites, such as EcoRI (AATT cohesive end) this is a bit high, but it still works. 10C would be better. I have yet to see a cohesive ligation that works overnight and does not work in 30 minutes. You can follow the success of ligations with different cohesive ends using the double length product trick. Warning -- heat killing ligase with PEG (quick ligases) will mess things up, both for your gels and for your transformations. You want to heat kill the ligase to make the gels run prettier.

#21 wizzkid

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Posted 12 March 2010 - 07:40 AM

prehaps the overnight digestion is the problem. Most restriction enzymes i use such as BamH1 only require a 2 hour incubation period at 37 degrees to digest samples efficiently. that could then reduce your background troubles :huh:

and for ligations i use T4 DNA ligase (NEB) and find it works best when you leave your sample at room temp for 2 hours

#22 jiajia1987

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Posted 15 March 2010 - 07:23 PM

Hi everyone, I am here to update on what I've done so far.

I started off with a fresh miniprep of pet22b vector, out of which I took 20ul for double digestion with Nde1 and BamH1(HF) since they are compatible in NEB4. Instead of digesting overnight, I did the digestion at 37degC for 2 hours. The reactions were cleaned up using a PCR cleanup kit since gel purification has been said to be able to inhibit ligation. A little of the cleanup was then used for a test ligation (just the cut vector alone without any inserts) with T4 DNA ligase.

I then took 50ng of the fresh miniprep, 50ng of the cleaned up double-digested pet22b vector and 50ng of the test ligation for transformation with HIT cells. I got a TONNE of colonies for the fresh miniprep, which was to be expected. The cleaned up double-digested pet22b vector had a number of colonies so a small fraction of the vector was uncut, which was fine with me. But the test ligation had the colonies filled up the plate, which was really depressing. This would show that single cut has been happening.

Phage434, I was bringing up your point of ligating single cut vectors to see if double fragments would appear. But, the effective concentration of the two compatible ends on the same linear single cut fragment is higher than two ends on two linear single cut fragments, isnt it? As a result, a majority of it would self-ligate instead of inter-ligate and we would get a circular product, which is the original fragment itself.

On one hand, I am wondering if I should move on to doing sequential digestion to see which enzyme is not cutting properly, but this would just waste even more time.
And Dukey and Denny... I think I am most probably moving on to finding another restriction site to go with Nde1. This has been wasting a lot of my time. As I am doing cloning of a mutated library, I will need double digested vector backbones.

I thank everyone for your ideas, suggestions and advices. They have definitely helped a lot and I will incorporate them in my work.

#23 jiajia1987

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Posted 15 March 2010 - 07:23 PM

prehaps the overnight digestion is the problem. Most restriction enzymes i use such as BamH1 only require a 2 hour incubation period at 37 degrees to digest samples efficiently. that could then reduce your background troubles :lol:

and for ligations i use T4 DNA ligase (NEB) and find it works best when you leave your sample at room temp for 2 hours


Hi wizzkid,

I did that. The ligation is working well. The problem lies with the digestion. =(

Edited by jiajia1987, 15 March 2010 - 07:24 PM.


#24 Dukey

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Posted 18 March 2010 - 09:50 AM

prehaps the overnight digestion is the problem. Most restriction enzymes i use such as BamH1 only require a 2 hour incubation period at 37 degrees to digest samples efficiently. that could then reduce your background troubles :lol:

and for ligations i use T4 DNA ligase (NEB) and find it works best when you leave your sample at room temp for 2 hours


Hi wizzkid,

I did that. The ligation is working well. The problem lies with the digestion. =(


The problem almost ALWAYS lies with the digestion, most specifically the second digest as this one you cannot monitor on the gel. That is where you need to focus your attention.

#25 perneseblue

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Posted 18 March 2010 - 04:14 PM

just a quick question, how far apart are the NdeI and BamHI sites? NdeI requires 8bp skirting the site for any efficiency at cutting.
May your PCR products be long, your protocols short and your boss on holiday

#26 jiajia1987

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Posted 18 March 2010 - 04:52 PM

just a quick question, how far apart are the NdeI and BamHI sites? NdeI requires 8bp skirting the site for any efficiency at cutting.


Hi pernesblue,

They are about 80+bp apart on the pet22b(+) vector.

#27 jiajia1987

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Posted 18 March 2010 - 10:11 PM

prehaps the overnight digestion is the problem. Most restriction enzymes i use such as BamH1 only require a 2 hour incubation period at 37 degrees to digest samples efficiently. that could then reduce your background troubles :)

and for ligations i use T4 DNA ligase (NEB) and find it works best when you leave your sample at room temp for 2 hours


Hi wizzkid,

I did that. The ligation is working well. The problem lies with the digestion. =(


The problem almost ALWAYS lies with the digestion, most specifically the second digest as this one you cannot monitor on the gel. That is where you need to focus your attention.


Hi Dukey,

Yes the problem lies with the digestion. A postdoc from my lab helped me to run a gel. I did sequential digestions with either enzymes eventually. Nde1 seems to be causing a problem as it has a very faint band corresponding to the main band that the undigested vector gave. BamH1(HF), on the other hand, gave a clean band.

#28 Dukey

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Posted 20 March 2010 - 05:07 PM

[/quote]

Hi Dukey,

Yes the problem lies with the digestion. A postdoc from my lab helped me to run a gel. I did sequential digestions with either enzymes eventually. Nde1 seems to be causing a problem as it has a very faint band corresponding to the main band that the undigested vector gave. BamH1(HF), on the other hand, gave a clean band.
[/quote]

I am not surprised, it only takes a very small amount of undigested vector to give HUGE background in your negative control plates. Most people always try to blame other factors when their cloning is not working, i.e. UV damage, ligation, star activity etc. In my experience it is almost never any of these things and the problem can always be traced back to digestion problems. Usually it is the PCR product which is to be inserted into the new vector which is the problem as PCR products do not digest particuarly well. This results in inefficient cloning and few positive colonies. But since you are working with a insert which has been cut from a vector, you can rule that out as if it was not cut 100% you would not have it to cut out.

Your problem is the enzyme combination for your plasmid, no question about it, or a dud enzyme batch (highly unlikely). I would have already switched enzymes to be honest. If you are determined to stick with it, cut the plasmid first with Nde1 and do not proceed until you have it digested. Stay away from double digests at the moment, that's just lazy when you have to troubleshoot. Also consider switching to Fermentas for enzymes, they are the highest grade in the industry. They have rapid 5 min enzymes that work brilliantly and literally will cut in a few minutes and if you were determiend to do double digests, they have a single buffer system.

Good luck.

#29 hanming86

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Posted 20 March 2010 - 09:10 PM

Try NdeI and XhoI if you could. works well. with promega both can work in Buffer D. essentially it should save you alot of work. and the efficiency is very nice too.
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#30 jiajia1987

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Posted 21 March 2010 - 07:23 PM

Try NdeI and XhoI if you could. works well. with promega both can work in Buffer D. essentially it should save you alot of work. and the efficiency is very nice too.



Hi Hanming,

I was thinking of using these two enzymes too! And both sites are present on pet22b(+)! :lol:




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