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Weird Library Transformation Results


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#1 Christos Karamitros Oyama

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Posted 10 August 2012 - 03:24 PM

Hi everybody,

I would like to describe you my problem I have been struggling with the last 1 month! I am trying to clone my gene (5 NNS) library into a pET28a vector. I have incorporated NcoI at the 5' and BamHI at the 3' of my PCR products. Just to mention , that I have generated my library using Overlap Extension PCR methodology because I wanted to randomize 5 codons in the middle of the gene. I obtain constantly my PCR product without any problems and background, at the exact and expected sequence length (~ 1 kb). I digested the vector overnight @ 37 C with the 2 different enzymes and afterwards I gel-purified it. I digested the PCR product (4 ug) for ~ 4 hours at 37 C after gel-purification and then I cleaned it up using a PCR clean up kit. I performed the ligation reaction following 2 different treatments : overnight @ 16C and at R.T. for ~ 2 hours. Ultimately I desalted my ligation product and I electroporated MC1061 EC (100 uL with 5 uL ligation mixture). In parallel, I prepared a negative control meaning digested vector incubated with T4 DNA ligase and buffer without insert. I transformed the negative control accordingly maintaining identical conditions.

Results next day:

Plate with negative control = 30 colonies

Plate with insert = "lawn" ~ 10^9 transformation efficiency! ( I have prepared reaaaally good EC using HPLC purified water!)

Up till here everything is OK. However, when I isolated plasmid DNA from 20 colonies and I sequenced them, I only got the vector sequence!!!! No insert at all!
How can it be? The negative control gave nothing regarding the background i.e. self-ligated vector. I am convinced that there is no problem with self ligation of the vector! After ligation, I run an aliquot on 1% agarose gel and I observed the characteristic supercoiled pattern of plasmid; implying that the ligation was successful.

I repeated the experiment changing different conditions such like incubation times (digestion, ligations e.t.c.), preparing fresh PCR products even! Again the same result! Negative control = 30-35 colonies and test sample = 10^9 cells and sequencing gave ONLY the vector; no insert!
If it were a self-ligation problem I would have observed that in case of negative control I assume.

Personally I do not have an explanation for that.

Any suggestion and opinion is mostly welcome!

Thank you in advance.

Chris

#2 leelee

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Posted 10 August 2012 - 05:20 PM

When you go about isolating your plasmid from your 20 clones, how do you do this?

Do you streak out some of your bacteria from the lawn and then chose colonies from that?

#3 Christos Karamitros Oyama

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Posted 10 August 2012 - 05:37 PM

Hi Veteran

and thanks a lot for your reply. Actually, on the plates there are individual colonies as well except from the "lawn" I mentioned. So I can isolate individual , well-defined and separate colonies. Additionally, what I did was to scrape some amount of the "lawn" and inoculate LB, from which I isolated plasmid DNA and ultimately I digested it with the 2 restriction enzymes (NcoI & BamHI) expecting to see the vector + the insert...But eventually nothing...Only linearized vector.

I read somewhere in another forum that replace of the used competent cells might help, meaning that the ones I am using are contaminated with uncleaved plasmid somehow but I think is quite unlikely since the negative control gives only 30 colonies which is within the acceptable limit of background.

Any ideas?

Once more, many thanks.


Chris

#4 phage434

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Posted 10 August 2012 - 06:47 PM

I'd want to run a negative control of the insert only, or the insert only ligated. You may not be fully removing template from the PCR reaction, or not amplifying the region of your template that you think. Any other differences between your experiment and negative control? Different outgrowth medium? Different plates? Have you checked that the plates are sterile? How are you desalting? Are you desalting the negative control?

#5 Christos Karamitros Oyama

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Posted 10 August 2012 - 06:56 PM

Hi phage434,

thank you very much fro your reply. Actually it is almost impossible not to have got rid of the template because it is an overlap extension PCR. That means that I run my first PCR and I got the first half of my gene. Then a second PCR gave me the second half of the gene and ultimately on the top of those 2 fragments which were combined I performed the last third overlap extension PCR with the 2 external wild type primers of the gene. The band I got is exactly the one I was expecting ~ 1 kb. So there are several steps till the final product which make the possibility of the initial template maintenance quite unlikely.

Negative control and my test samples were treated identically in both trials. I desalted them using a spin column for PCR product clean up and I eluted with water. I also desalted the negative control.

I have cloned many similar libraries without any single problem. But this time I am really frustrated because I have ran out of ideas anymore I do not want what to think.....

Thanks a lot,


Chris

#6 leelee

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Posted 10 August 2012 - 06:58 PM

So you have larger "single" colonies on top of a finer lawn? Or is there clear agar around them?

And for future applications, it isn't advisable to take a scrape of a lawn of bacteria and try to purify plasmid from it. Especially when it is a transformation plate (rather than a plate from a pure culture). This scrape would have contained many many different clones, so it would be mixture and therefore completely useless to you.

How much kanamycin are you adding to your plates? (final concentration).

Also, I agree with phage34 in the points he has made for you to consider. The contamination of the insert with template seems like a good explanation for what you are seeing.

#7 Christos Karamitros Oyama

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Posted 10 August 2012 - 07:06 PM

Hi leelee,

no, the colonies I picked were surrounded by agar and they were far away from the lawn. They were individual independent colonies.

I did scrape some of the lawn because I wanted to get a picture of what is the percentage of the background in my library. This is exactly what I wanted to see: how different are the clones, meaning how many carry the insert and how many do not; and apparently the insert was not present anywhere because after digestion I did not see my insert but only linearized vector.

I am using final concentration of kanamycin 50 ug/ml.

I understand guys what you are saying about contamination but I cannot imagine from where! It sounds that I have to throw away everything and start from the beginning.!!!


Chris

#8 leelee

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Posted 10 August 2012 - 07:23 PM

I still think picking colonies from a plate that has a lawn is dubious.

Presumably you spread plated? Yes? So then if there is a visible lawn at one part of the plate and not another? How does that happen?

It might be worth streaking some colonies out and picking from the streak plate- even just do a quick colony PCR for your insert to screen a whole bunch at once without having to purify so much plasmid.


I was thinking too, if what you are seeing was from template contamination, obviously your sequencing results would come back as rubbish because your primers will be specific for the vector and not the template.

And this is a stupid question, so forgive me, but you do use the same aliquot of digested vector to make both your +ve and -ve ligation mixes, yes?

Hmmmm

I think you are right and your best bet is to start again. It will probably take just as much (if not more) time to figure out what is going wrong here- and hopefully if you do it again, it will work this time!

#9 Christos Karamitros Oyama

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Posted 10 August 2012 - 07:36 PM

Hi again leelee,

once more many thanks for your reply. Look, with my transformation volume (1 ml SOC), I streaked 8 squared Corning plates and in some of them I got lawn while in some others I used less volume and I only got well distributed colonies. So, I selected individual colonies from those plates which showed spatial growth. I also performed just today some colony PCRs with 15 additional clones from other plates and still nothing!

Do not say your question is stupid, I am also sure I have done something stupid and I get what I get. Yes, I used the same vector batch for both the +/- controls.

I think I will try for 3nd successive time and hopefully it will work. If not, I will bother you again. But please tell me that this result is very odd! I am not the only one who thinks that....

Many thanks again....

#10 ascacioc

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Posted 10 August 2012 - 10:56 PM

Hi Chris,

I have some suggestions that might not solve your problems but ease the finding of a solution:
-I know that you are explaining above that you do not have problems with initial template in the overlap PCR, but just for the 3rd repetition to be on the safe side: since you do not do the gel extraction of the PCR product, you can add an overnight digestion step with DpnI of the final PCR product just before PCR clean-up
-check whether your competent cells are not contaminated with smth resistant to Kan i.e. streak a bit of them on a Kan agar plate. I know that it sounds imposible and this probably never happened to you, but last time I was so happy about my competent cells as you are in this case, I had a special variant of DH5alpha (gotten from other people) that happened to be Kan resistant. It might be the case that you really do have a contamination in the competent cells. It happens to the best of us:)
-when you do the transformation and plating, maybe you do dilution plates: 10 uL transformant, 100 uL transformant and the rest to not have the lawn anymore
-I have read here on the forum these days a great advice for getting read of religations: digest your ligated reaction with another restriction enzyme from the MCS that should have been removed from the MCS by the insertion of your gene; this is how you get rid of the empty vector before transformation i.e. inside the MCS --R1---R3---R2-- you used R1 and R2 for cloning; R3 (that is not also in your insert, check before for this) should not be anymore in the final plasmid, only in the relagation --> use R3 to linearize empty vector.

Hope this helps, at least the last advice,

Andreea

#11 Christos Karamitros Oyama

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Posted 11 August 2012 - 06:21 AM

Hi Andreea,

thank you very much for your advice. I will definitely consider your suggestions during my 3nd successive trial of cloning. Let's hope that this will work this time.....

Best,

Chris




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