Colonies without inserts - (Feb/03/2011 )

Hello, my name is Claudia and I am from Malaysia

I am having trouble with my ligation (or so I think). Once I have ligated at 16 degrees and left it overnight, colonies are formed on the plate (LB + ampicillin). However after running colony PCR, my insert is not to be found. I am using the pGEMT system. I am using a 1:8 ratio. 0.5ul pGEMT, 0.5ul ligase, 5ul 2x buffer and 4ul insert. I transform 10ul of ligation product into 100ul of TOP 10 E.coli competent cells. The cells are fine because I tried it out with the pRSET vector and the next day the LB ampicillin plate was filled with colonies

Could it be a ligase problem? I understand that the pGEMT vector is linearized and so if colonies are present, those cannot be the unlinearized vectors, right? I am using M13 primers when doing the colony PCR and as such after I have viewed the gel there are only bands present at ~250bp.

I am in dire need of help and any form of suggestions is highly appreciated

Thank you

Claudia

With your 2x buffer, I'm guessing you are using quick ligase. I would switch to normal ligase buffer, and make sure that the 10x buffer is not too old (or add ATP as a supplement). Is your insert prepared with a Taq pcr enzyme? It must be, to add the A overhangs necessary for T ligation. I believe you are transforming too much ligation product -- I would transform only 2-4 ul of the ligation. More can inhibit transformation.

Hi,

Thank you for your reply. The 2x buffer and ligase come together in the pGEMT set and so that's the one I am using, Yes my insert is prepared with Taq enzyme.

As for using too much ligation product, I suppose that could be possibility but this has been done in my lab many times and there appeared to be no problems. I have also tried it before without facing any problems. It just doesn't seem to be working now

Claudia

Claudia88 on Fri Feb 4 02:18:21 2011 said:

Hi Claudia,

What about the 1:8 ratio of pGEM-T to insert? Can you try changing that ratio?

I doubt temperature has much to do with it, but what a tech used to do in a place I worked, which I've carried on doing is put the ligation tube floating in room temperature water, and put that in a fridge overnight, so it starts off at room temperature, and the temperature decreases. IIRC, the protocol advises either room temperature for an hour or 4 C overnight, right? Still, maybe the ratio is a better bet.

Hi Sean,

Thank you for your reply.

Alright, I will try playing around with the vector:insert ratio. But might there be any other possible reasons as to why this is happening?Perhaps there are some information that I may have left out when posing my question. Do you have any queries?Perhaps that would better enable you to guide me

Thanks again!

Claudia

Claudia88 on Fri Feb 4 13:21:33 2011 said:

Hi Claudia,

I'm a bit of a noob myself, so there's not a lot of wisdom in me, yet! Did you try to do blue/white screening by applying X-Gal and IPTG to your plates? Also, the pGEM-T kit comes with a control insert - you might try running that alongside your experimental ligations, using the manufacturer's advised ratio. You need to ensure a good homogenous spread of IPTG/X-Gal to make sure you can trust in the blue/white screen. I did it recently, and my colonies were too dense and the colouration was difficult to detect so the blue/white screen was virtually useless, but I was lucky enough to get 19 out of 20 colonies with insert!

Other than that, I can't really advise anything else.

I have the same problem. The transformation is successful, but there is no right insertion. Below is my post also. If you have solved the problem, please let me know. Thanks.

I am trying to clone a 1,200 bp PCR products of PISTILLATA in the plants. I did directly clone the PCR products. The half of the clones was white. When I check the size of the insert by PCR amplification, there was no right insert (50-100 bs of inserts). I ran the PCR products on the gel, and cleaned the right fragment from the gel. This time, the cloning did not work at all. Only 2-4 blue clones, nothing more on the plate. I did this test several times, but no success. Anybody can suggest any solution? Thank you.

I am also trying to clone insert size of 1.1 kb into 5.5 kb vector. we tried with enzyme EcorI and Hind III and alternatively with Bam HI and Xho I, but I am not able to get the insert. We do elution after cutting the gel and do quantification of both insert and vector and I use 1:3 concentration ligation. But I am not able to get the clone. Anybody suggest me solutions on this matter.

If the method of transformation was electroporation, some ligase kits (eg. Fermentas) recommend to purify the DNA (ligation mix) before transformation to increase the transformation efficiency. Will the problem be your colony PCR conditions? A 94deg/15 min step will be necessary, the bacterial cells have to break and the DNA has to be denatured.

Deepak, do you purify the products between digestions? For example, it will be good to PCR purify (using a kit) your product after Ist enzyme digestion, then digest with the second enzyme, then PCR purify the product after the 2nd digestion, then use it for ligation. It works for me. Also, when gel extracting, avoid too much of UV exposure.

Are you getting colonies, but without the inserts? We need to know many more details about how you are doing these reactions. In general failures in ligation are due to transformation, not ligation. How are you transforming? How competent are your cells?