ligation troubleshooting - Trouble in inserting purified PCR product into pFlagCMV (Jul/24/2009 )

Hello,

I have been trying for 3 months to insert my pcr product(2kb approx.) into pFlagCMV. First of all i have successfully inserted my pcr product into pGEMT cloning vector and sequenced it correctly. Now when i am trying to cut the pcr product from it and insert it into pFLag i am having the trouble. I have tried ECOR1 and Sal1 digestions for one month and have not found any colony after trasnformation with highly competent DH5alpha cells. I have tried in-gel ligation, ethanol precipitaion, gel extraction. But nothing helped.

Recently i add ECOR1 sites to my pcr product by doing pcr from the pGEMT plasmid(containing my insert). It was successful and i also gel purified my ECOR1 site containing pcr product with Quagen quickgel extraction kit. After running gel i saw my desired band. But again when i tried to ligate the purifed pcr product with pFlagCMV after digesting both insert and vector with ECOR1, i don't find any colony both in my control (vector alone) and insert+vector.

I am frustated now. please help

We need much more detail. How are you cleaning up your pcr reaction? How are you digesting? Are you gel purifying? How much dna are you ligating? How are you ligating? How are you transforming? What controls do you have? We can't begin to debug this until we know these things.

It amazes me that people think we can help with so little information.

phage434 on Jul 24 2009, 08:29 PM said:

i think i have mentioned in my previous post that i am extracting my pcr product from the gel and cleaning it throuch quagen quick gel extraction kit which cleans and purify DNA. I am digesting it with ECOR1 in 20 ul reaction which has 1ul of ECOR1 and 2ul of 10x buffer (h) plus my DNA and DI water. i am ligating with T4 DNA ligase (1ul) with 2(ul) of ligase buffer and vector+insert in (3:1) ratio in a 20ul reaction.
I have only i control which is vector alone (that has been dephosphorylated and restriction digested as the vector in vector +insert but doesnot have the added insert in it)
I think i have mentioned earlier that i am transforming with highly competent DH5alpha cells which comes as 50ul vials and i am adding 2ul of my ligation reaction in it.

One more thing the pFlag CMV2 that i am using is not store bought, i have done large scale prep and made it from the store bought vial when it got finished. Its concentration is 375ug/ml. the concentration of my pcr product i don't know as i have not measured it.
Am i having a problem because of using unknown concentration of my purified pcr product and the handmade pFLagCMV? What do u recomend?

Some things that could be problems:
* You could be damaging your DNA during gel isolation with UV. Make sure you use brief exposure to long wave (365 nm) UV or better, blue light, to find your fragments. Others have suggested 1 mM guanine in the gel to protect the DNA, but I have no experience with this. Do you really need to gel extract? If you have a single PCR band, try just PCR purification followed by cutting, then directly into the ligation.

* Do you heat kill the EcoRI to inactivate it prior to the ligation? Active RE will just re-cut ligated fragments.

* How are you dephosphorylating your vector? I would try to eliminate this step through redesign using two-enyzme ligation, but if you are doing it, make sure you use minimal amounts of enzyme for minimal time, and remember to inactivate it. SAP or Antarctic phosphatase can be heat killed. Over digestion can damage the cut ends.

* How much DNA are you adding to your ligation reaction? There is a tendency to think that more is better, but this is not true. You want intra-molecular reactions for closing the circular plasmid, and these are favored at low concentrations. Something like 20 ng of DNA in a 10 ul reaction is a good place to start. This will also help by diluting inhibitors that might be present in your DNA prep. Make sure your ligase buffer (not only the ligase!) is fresh. It contains ATP and will degrade with heat/thaw cycles. Room temperature ligation for 30 minutes is usually enough. If you in a hot location, you might need to lower this a bit and extend the time.

* I would definitely measure my DNA concentration following the PCR cleanup. You need to know if there is some DNA left. Ideally, run a gel and compare band size and strength.

* Usually ligation problems are really DNA preparation problems. Get the DNA under control, and the ligation is easy.

* How do you know your cells are "highly competent." You need to measure this, since cells can start competent and be damaged by most anything. Do 10x dilutions of a know plasmid (pUC19 e.g.) at a known concentration, transform, and measure the competence (usually in units of CFU/ug of plasmid DNA). Something like 10 pg/ul is a good concentration to transform, adding 1 ul to your cells. At least 10^8 cfu/ug should be used for these ligations, though you might get by with a little lower. If you use your uncut plasmid as this control, you will also know you have the antibiotics and growth conditions correct.

* I'd be surprised if EcoRI was the only enzyme in common between these vectors. If you have two that flank your insert and are spaced sufficiently far apart in your target vector, consider a double digestion of both, followed by a ligation and transformation. You get orientation control with this approach, and may be able to eliminate the dephosphorylation step and gel purification steps. In general, the less you do to your DNA, the better.

Hey everyone !

Thanks for all your suggestions..I have successfully ligated my insert with the vector