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Struggling for months with one little ligation/transformation - please help!


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#1 Hopeless

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Posted 29 June 2009 - 06:52 AM

Hello everybody!
I have been reading the topics in this forum now for a while and discovered the expertise and wisdom that is here. You are the ones I need to ask.

I have a problem. ;)

I have been struggling now already for quite some time with one lousy transformation (of my bait plasmid). And actually this is supposed to be only the first step of my Y2H screen I'm actually supposed to do (and I only have time until the end of this year since I'm doing my master thesis)...

This is the case: I have an 1,5 kb insert (Arabidopsis protein phosphatase 2A subunit B ) which has already been subcloned into pDrive and succesfully cut out of there with XhoI (I'm using single digestion). Now I need to clone it into the 4,8kb vector pHybLex/Zeo (Invitrogen).
This I have nicely linearised and desphophorylated with the anarctic phosphatase by NEB (no colonies on religation control plate).
I have tried the following ligation reactions and conditions:
- 10ng vector/100ng insert --> 4degrees celcius o/n,
- 10ng vector/60ng insert --> 4 degrees o/n,
- 100ng v/50ng i --> 4 degrees o/n,
All of these ligations resulted some colonies after transformation into Dh5alpha E.coli. But each time they were not containing an insert (mini test-digested with XhoI).
I also tried some other ligations conditions like 16 degrees and RT over night...they did not bring any colonies.
We are using T4-ligase from NEB.

I actually don't understand what is going wrong. Since the vector is dephosphorylated I would expect no colonies to appear if the insert is not in the vector. Still always some negative colonies appear.

I would appreciate every kind of help you can give me...what can I do differently? Does somebody have experience with the pHybLex/Zeo plasmid?

Kind regards,
Hopeless

#2 Michaelro

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Posted 29 June 2009 - 11:05 AM

Hello everybody!
I have been reading the topics in this forum now for a while and discovered the expertise and wisdom that is here. You are the ones I need to ask.

I have a problem. ;)

I have been struggling now already for quite some time with one lousy transformation (of my bait plasmid). And actually this is supposed to be only the first step of my Y2H screen I'm actually supposed to do (and I only have time until the end of this year since I'm doing my master thesis)...

This is the case: I have an 1,5 kb insert (Arabidopsis protein phosphatase 2A subunit B ) which has already been subcloned into pDrive and succesfully cut out of there with XhoI (I'm using single digestion). Now I need to clone it into the 4,8kb vector pHybLex/Zeo (Invitrogen).
This I have nicely linearised and desphophorylated with the anarctic phosphatase by NEB (no colonies on religation control plate).
I have tried the following ligation reactions and conditions:
- 10ng vector/100ng insert --> 4degrees celcius o/n,
- 10ng vector/60ng insert --> 4 degrees o/n,
- 100ng v/50ng i --> 4 degrees o/n,
All of these ligations resulted some colonies after transformation into Dh5alpha E.coli. But each time they were not containing an insert (mini test-digested with XhoI).
I also tried some other ligations conditions like 16 degrees and RT over night...they did not bring any colonies.
We are using T4-ligase from NEB.

I actually don't understand what is going wrong. Since the vector is dephosphorylated I would expect no colonies to appear if the insert is not in the vector. Still always some negative colonies appear.

I would appreciate every kind of help you can give me...what can I do differently? Does somebody have experience with the pHybLex/Zeo plasmid?

Kind regards,
Hopeless


Just to make sure that You use low salt LB for the LB-Agar plates preparation.
Following transformation, the bacteria shoud be grown on low salt LB-Agar with Zeocin.
Otherwise it is problematic to obtain the resistant colonies.
Also maybe You should try another ligation kit such as Rapid Ligation kit from Roche
Good Luck

#3 positivecontrol

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Posted 29 June 2009 - 11:47 AM

Hello everybody!
I have been reading the topics in this forum now for a while and discovered the expertise and wisdom that is here. You are the ones I need to ask.

I have a problem. ;)

I have been struggling now already for quite some time with one lousy transformation (of my bait plasmid). And actually this is supposed to be only the first step of my Y2H screen I'm actually supposed to do (and I only have time until the end of this year since I'm doing my master thesis)...

This is the case: I have an 1,5 kb insert (Arabidopsis protein phosphatase 2A subunit B ) which has already been subcloned into pDrive and succesfully cut out of there with XhoI (I'm using single digestion). Now I need to clone it into the 4,8kb vector pHybLex/Zeo (Invitrogen).
This I have nicely linearised and desphophorylated with the anarctic phosphatase by NEB (no colonies on religation control plate).
I have tried the following ligation reactions and conditions:
- 10ng vector/100ng insert --> 4degrees celcius o/n,
- 10ng vector/60ng insert --> 4 degrees o/n,
- 100ng v/50ng i --> 4 degrees o/n,
All of these ligations resulted some colonies after transformation into Dh5alpha E.coli. But each time they were not containing an insert (mini test-digested with XhoI).
I also tried some other ligations conditions like 16 degrees and RT over night...they did not bring any colonies.
We are using T4-ligase from NEB.

I actually don't understand what is going wrong. Since the vector is dephosphorylated I would expect no colonies to appear if the insert is not in the vector. Still always some negative colonies appear.

I would appreciate every kind of help you can give me...what can I do differently? Does somebody have experience with the pHybLex/Zeo plasmid?

Kind regards,
Hopeless



Not sure why your re-ligation negative control plate had no colonies; if you're getting empty vector colonies otherwise, it clearly should have.

Anyway, try these things if you haven't already: Linearize your vector with an overnight digest, gel purify both the vector and the insert after cutting, and heat inactivate the phosphatase. Replace your ligase buffer (it contains ATP, which goes bad after many thaws) and probably your phosphatase too (doesn't seem to be working very well). I always ligate @16 C o/n.

And don't lose hope!

#4 Hopeless

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Posted 29 June 2009 - 12:13 PM

Just to make sure that You use low salt LB for the LB-Agar plates preparation.
Following transformation, the bacteria shoud be grown on low salt LB-Agar with Zeocin.


Yes. I do...:)...have you worked with this plasmid?

Anyway, try these things if you haven't already: Linearize your vector with an overnight digest, gel purify both the vector and the insert after cutting, and heat inactivate the phosphatase. Replace your ligase buffer (it contains ATP, which goes bad after many thaws) and probably your phosphatase too (doesn't seem to be working very well).


The linerization worked well....I purified the vector and also the inserts from a gel. After gel elution I desphosphorylated (only vector ;) ) and also deactivated at 65 degress for 20 min (its a brand new phosphatase - should work fine). Also ligase and ligase buffer are new...ligase buffer has just been thawed once, when I took aliquots which I then thaw when needed.

You see why I don't know what to do....it seems to me that I have already done quite alot....

But many thanks for the suggestions and please let me know if you have any other ideas!! :)

#5 positivecontrol

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Posted 30 June 2009 - 04:33 AM

Just to make sure that You use low salt LB for the LB-Agar plates preparation.
Following transformation, the bacteria shoud be grown on low salt LB-Agar with Zeocin.


Yes. I do...:)...have you worked with this plasmid?

Anyway, try these things if you haven't already: Linearize your vector with an overnight digest, gel purify both the vector and the insert after cutting, and heat inactivate the phosphatase. Replace your ligase buffer (it contains ATP, which goes bad after many thaws) and probably your phosphatase too (doesn't seem to be working very well).


The linerization worked well....I purified the vector and also the inserts from a gel. After gel elution I desphosphorylated (only vector :P ) and also deactivated at 65 degress for 20 min (its a brand new phosphatase - should work fine). Also ligase and ligase buffer are new...ligase buffer has just been thawed once, when I took aliquots which I then thaw when needed.

You see why I don't know what to do....it seems to me that I have already done quite alot....

But many thanks for the suggestions and please let me know if you have any other ideas!! :)


Ah, you do have a sticky problem.

This solution might seem a little strange, but empirically it worked when I had a similar problem. After you gel purify the insert, concentrate it by ethanol precipitation and bring it up in 10 uL TE. (I am assuming you use a kit to gel purify). Don't ask me why concentrating a Qiagen kit eluate of 30 uL to 10 uL by ethanol precipitation would make much difference, but it really did for me. (Perhaps it's an issue with salts being eluted with the DNA ... I don't know.)

Stick with the higher insert:vector ratios for the ligation (I usually calculate the ratio based on moles rather than mass; I would try 10:1 5:1 and 2:1 molar ratios). You might end up getting a lot of clones with more than one insert, but at least that's headed in the right direction.

Also, screen lots and lots of clones, if you have them. With any cloning project I do now that doesn't work right away, I screen 48 clones at a time, as long as I get that many (48 because benchtop microcentrifuges hold 24 miniprep columns, and screening 2 rounds of 24 minipreps seems to fit in one day of work). One time, I got a single clone out of 48 that was exactly right. And it only takes one...

It's not very elegant, but sometimes brute force will do it.

#6 Hopeless

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Posted 30 June 2009 - 05:24 AM

This solution might seem a little strange, but empirically it worked when I had a similar problem. After you gel purify the insert, concentrate it by ethanol precipitation and bring it up in 10 uL TE. (I am assuming you use a kit to gel purify). Don't ask me why concentrating a Qiagen kit eluate of 30 uL to 10 uL by ethanol precipitation would make much difference, but it really did for me. (Perhaps it's an issue with salts being eluted with the DNA ... I don't know.)


Thanks alot...I think I will try this...I'm actually today starting to do more insert, so that at the same time I also can try this method of yours! :) I'm using the gel elution kit by macherey&nagel....

Stick with the higher insert:vector ratios for the ligation (I usually calculate the ratio based on moles rather than mass; I would try 10:1 5:1 and 2:1 molar ratios). You might end up getting a lot of clones with more than one insert, but at least that's headed in the right direction.


That is a really good point...I haven't been thinking about that yet. But please help a dumb girl...is there somehow a programm that can calculate the molar mass of my insert/vector according to their sequence or do I just have to do that on my own....because then I will also definitely try to calculate the ratios new! By the way...found this equation somewhere on the net....what do you think about it?
insert mass in ng=6*[insert length in bp/vector length in bp]*vector mass in ng (here for a ratio of 1:6)...is this already going into the direction of molar ratios?

Also, screen lots and lots of clones, if you have them.


Yeah well I have actually been screening all of the clones i so far have got - they just have not yet been that many :/....
What do you think about colony pcr - during subcloning it helped me to screen masses of colonies....worked quite well almost every time...

Next question: what do you think about purifying the ligation (afterwards) by PCR purification kit? I was recommended to do that today....

Again, thank you all for your ideas!! :P

#7 HomeBrew

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Posted 30 June 2009 - 07:50 AM

Frequently, problems of this sort are caused by the vector ends being damaged by the dephosphorylase. Dephosphorylation is much more tricky than most people realize...

When I dephosphorylate a vector, I'll add 1/5th to 1/10th the recommended amount of CIAP at the end of the restriction digest, incubate at 37C for 3 to 5 minutes, then pull the sample from the waterbath and load it directly on a waiting gel for gel purification. Waste no time, work quickly, and turn your gel on immediately.

We also add guanosine (0.28 g/L in 1x TAE, stir with gentle heat to dissolve) to the TAE used to cast and run a gel from which we are going to recover fragments. This acts as a UV protectant.

I find these two tweaks to be orders of magnitude more important to a successful cloning experiment than vector-to-insert ratios, which we rarely bother to calculate, and just estimate based on the amount of vector and insert seen on the purification gel.

Another thing we do is use Ready-To-Go T4 DNA Ligase and incubate at 16C in a PCR machine; I routinely go overnight on this incubation, but others in the lab incubate for shorter periods of time. The Ready-To-Go T4 DNA Ligase is a glassified mixture of T4, ATP, and buffer, and it is stable at room temperature. Use of this product eliminates variability due to ATP degradation from freeze-thaw cycles.

The final thing is that we use chemically-competent cells, usually DH5alpha, prepared by the RbCl2 method (see here).

We used to have many of the problems I see over and over here on the forums regarding cloning, but since we tweaked our standard protocol using the above things -- something we did years ago -- failures in cloning experiments are the exception, not the norm...

#8 Hopeless

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Posted 30 June 2009 - 08:07 AM

When I dephosphorylate a vector, I'll add 1/5th to 1/10th the recommended amount of CIAP at the end of the restriction digest, incubate at 37C for 3 to 5 minutes, then pull the sample from the waterbath and load it directly on a waiting gel for gel purification. Waste no time, work quickly, and turn your gel on immediately.


I use the Anarctic phosphatase from NEB. The advantage of this phosphatase is that it can be deactivated by heat. So therefore I dephosphorylate with the anarctic phosphatase for 30 min at 37 Degrees and after that I directly inactivate it at 65 degrees for 5 min and according to NEB there is now no need for a purification by a gel....
But still - I thank you for this information and I will try a ligation without the dephoshorylation step....

We also add guanosine (0.28 g/L in 1x TAE, stir with gentle heat to dissolve) to the TAE used to cast and run a gel from which we are going to recover fragments. This acts as a UV protectant.


This is really interesting...this I will also try....(it can't harm to try :P )

#9 fishdoc

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Posted 30 June 2009 - 08:09 AM

I had a problem with cloning awhile back. I was cloning into a suicide vector that required lambda pir, and the E. coli strain I was using was SM10 1-lambda pir. However, when I made my electrocompetent cells, I accidentally used SM10 cells without the lambda pir. I didn't realize that at first, and tried all sorts of ways to optimize the ligation when it was really the E. coli strain was the problem. I don't really have anything to add to the methods to make sure your ligation is working. Just wanted to say to make sure your E. coli strain contains the necessary components to allow your vector to replicate.

One thing you might try to ensure your insert is going into your vector is to do a PCR using your ligation product as template. Choose primers just outside your cloning region, and amplify across to see if your insert is going in. That may help to tell you if it's the ligation step that is a problem or something downstream.

As for calculating molar ratios for ligations, I use this site:

http://www.promega.c...ath/Default.htm

Good luck.

#10 HomeBrew

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Posted 30 June 2009 - 09:29 AM

I use the Anarctic phosphatase from NEB. The advantage of this phosphatase is that it can be deactivated by heat. So therefore I dephosphorylate with the anarctic phosphatase for 30 min at 37 Degrees and after that I directly inactivate it at 65 degrees for 5 min and according to NEB there is now no need for a purification by a gel....


The key points in my discussion of dephosphorylation is not the method of inactivation, but the amount of enzyme used (much less than is recommended) and the incubation time (also much less than is recommended). Heat inactivation is not an instantaneous thing -- I worry about what's happening to my vector ends as the sample temperature increases from 37C to 65C...

We gel purify everything. The amount of time spent doing this is more than made up for by getting the clone the first time, thus not needing to repeat the experiment many times...

This is really interesting...this I will also try....(it can't harm to try :) )


The "guanosine as a UV protectant" reference is Grungermann, D., and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21(5):898-903. The abstract is on PubMed (PMID 8922632), and, if you don't have an account with BioTechniques, there's a pdf copy here. We've been using this for a long time -- since it first came out in 1996, in fact.

In the paper, they note that the addition of 1 mM guanosine to the gel and the running buffer "increased the yield of clones by a factor of about 400, compared with conventionally prepared, unprotected DNA exposed to 312 nm UV". I've often thought the simple method outlined in this paper is under-appreciated and under-utilized.

#11 DrChicago

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Posted 21 July 2009 - 03:47 PM

Hey,
i hope youve found an answer to your question.
I was wondering where you found the pHybLex/Zeo vector... I was trying to find it and Invitrogen said they dont sell it anymore.
Could you please let me know where i can find it? If you just had it in your lab, maybe i could contact someone and ask for it?
I would appreciate any info.

Thanks,
Tassos
akonstan@medicine.bsd.uchicago.edu

#12 JeremyJ

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Posted 12 January 2010 - 12:25 PM

Hey,

Hope you've solved your problem by now? Was it the E. coli strain? Did you have "wrong" DH5?

from Invitrogen:
"
Any E. coli strain that contains the complete Tn5
transposable element (i.e. DH5αFIQ, SURE, SURE2) encodes
the ble (bleomycin) resistance gene. These strains will confer
resistance to Zeocin. For the most efficient selection, use an
E. coli strain that does not contain the Tn5 gene (i.e. TOP10,
DH5, DH10, etc.).
"




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