Transformation problem. Please help! - 1.7kb insert to 5.4kb vector; double digested w/ EcoRV & XhoI (Aug/04/2009 )

little mouse on Aug 6 2009, 11:14 PM said:

Sorry to reply late. I was told to do a in situ hybridisation during the past week.

I can confirm that right antibiotic is used since my colleague prepared the agar plates with me
I'm not pretty sure about the control you mentioned but the ligase used was newly arrived in late July and I think there shouldn't be any problem.

Ayukawa on Aug 17 2009, 02:57 AM said:

Hi.
I'm not sure if your DNA was dissolved in TE buffer. If yes, then probably you should just dissolve the DNA in sterile distilled water. The presence of EDTA in TE buffer might interfere with your ligation.

Also, have you tried the ratio of 1 vector to 1 insert?

Good luck.

Cheers.

These problems are almost always problems with DNA preparation or transformation, and rarely with ligation.

Tell us in detail how you are cutting and purifying your DNA.

Tell us what concentration of DNA you are using in your ligation, in what volume.

Tell us what controls you have done to confirm that your transformation efficiency is high.

I'll try to provide as many details as I can.

Source
Insert (1741bp)
50ul of insert was obtained through PCR amplification (from originally 1ul of DNA at a concentration of 1ug/ul) using PfuTurbo DNA Polymerase
The PCR product was purified using QIAquick PCR purification kit; 50ul of EB buffer was used for DNA elution

Vector (5.4kbp)
The vector used was pcDNA3.0-FLAG.

Digestion (insert)
Enzyme: XhoI(3ul) + EcoRV(3ul)
Buffer: 10x Tango (10ul, final conc = 2x as recommended by Fermentas)
DNA: 30ul
dH2O: 4ul to make up a final volume of 50ul

Digestion (vector)
Enzyme: XhoI(3ul) + EcoRV(3ul)
Buffer: 10x Tango (6ul, final conc = 2x as recommended by Fermentas)
DNA: pcDNA3.0-FLAG (5ul @ 1ug/ul app.)
dH2O: 13ul to make up a final volume of 30ul

*Both insert & vector were incubated overnight @ 37 degC waterbath

Gel excision
-Both insert and vector were cut under low power UV illuminator using DNase-free blade

DNA Extraction (Fermentas DNA Extraction Kit) (same for insert and vector)
The excised gel were weighed and required volume of binding solution(4.5x) & TBE conversion buffer(0.5x) were added accordingly (0.01g = 10ul app. as recommended by Fermentas DNA extraction kit).
-The mixture (gel+binding solutions+TBE conversion buffer) was incubated @ 55 degC for 5min (w/ mixing in every 2min)
-After the gel was completely melted, 10ul of silica suspension was added (to bind DNA), mixed and incubated @ 55degC (w/ mixing in every 2min)
-The silica was pelleted using table centrifuge, supernatant was removed thereafter.
-The pellet was completely resuspended in prepared ice cold washing solution. Again, the silica was pelleted again to remove supernatant. This was done 3 times according to Fermentas protocol.
-After the last wash, the pellet was air-dried.
-10ul of TE buffer was used to dissolve DNA. The pellet was resuspended and incubate @ 55degC for 5min. Then, the silica was pelleted and supernatant containing DNA was extracted. (This step was repeated 3 times)

With all steps described above, I obtained the digested insert and vector.

Ligation
As mentioned, I tried different ins:vec ratios (5:1, 4:1, 3:1), different incubation time (5min, 30min, overnight) with 2 different ligation kits
Kit 1 - Takara DNA Ligation Kit (Takara Mighty Mix)
- Equivalent amount of Takara Mighty Mix to that of insert+vector was added and incubated @ 16 degC, 30min

Kit 2 - Fermentas Ligation Kit (T4 DNA Ligase) (newly purchased)
- Insert+vector (volume 1)
- 5x Ligation buffer (4ul, final 1x)
- T4 DNA Ligase (1ul)
- dH2O (volume 2) was added to make up a final volume of 20ul
- incubate @ 22 degC, 5min (tried 16 degC overnight using this kit)

Transformation using DH5alpha.

With Takara kit, I managed to get colonies w/o insert. I can get no colony using Fermentas' so far.

Thanks.

Best,
Ayu

Hi read about your problem and have a few options:

Always ligate a positive control (cut with one enzyme only), Always ligate a negative control (double cut and w/o insert added) and a number of ratio mixtures with the vector and insert together.
Ligate the mixtures at ~16°C (normally I place the tubes at the end of the day in room temperature water in the refrigerator) overnight!

You can add an transformation enhancer like TRAEN if you want and transform 2-10ng in competent DH5alpha.
Control the amount antibiotics in the agar before plating! Grow the transformants overnight at 35°C and the next day small colonies will appear (you can set the temperature to 37°C until the colonies are big enough)

Good luck! Greetz memo

Some comments:

* I assume you have checked that there is sufficient 5' overhang on your PCR primers to allow the DNA to be cut with your enzymes. Bases in your primer are cheap.

* That's a lot of template for a PCR reaction. With 1 ug of template DNA, your amplified product will only be a few times more concentrated than the template DNA. Try to amplify with much less template to reduce the junk DNA in your final reaction.

* The DNA concentration in your restriction digests is way too high. Typically you want 1 ug in a 50 ul volume. You have 5 ug in a 30 ul volume. Also, I would not be using that much enzyme. Try 1 ul. Overnight is unnecessary and possibly damaging to your DNA. 1 ul of enzyme will have 20 units or so, enough to cut the 1 ug of DNA you should be using in your reaction in less than an hour at 37C. Heat kill for 20 minutes at 80C. You can conveniently do both in a PCR cycler.

At this point, I would go directly to a ligation. Too much handling of DNA is difficult and often damaging. If you have a good PCR band with few side products, and good digestion of your backbone, this ligation should be easy. As memo suggested, control digestions of your vector with each enzyme will make sure things are cutting. Use the gel to evaluate relative amounts of insert/vector. Remember that the ideal spot is 1-3x as many molecules of insert as vector (molar ratio), but people tend to worry too much about this.

The blunt ligation is much more difficult than a cohesive end ligation. I would probably try it with a PEG containing buffer ("quick ligase.") What I would really do is avoid having to do a blunt ligation. Overnight, as suggested, at low temperature. You don't mention the amounts of DNA in your ligation. You are probably using too much DNA. I would try 20 ng of vector and the correct amount of insert by molar ratio, likely around 10-20 ng.

You say very little about your transformation protocol. In particular, you should add a maximum of 2 ul of ligation mix to the competent cells. If you are adding 20 ul of ligation product to 50 ul of cells, you will have very low efficiency transformation. I STRONGLY suggest doing a control transformation with 10 pg of pUC19 DNA (or some other convenient plasmid) to establish the competency of your cells.

phage434 on Aug 18 2009, 09:55 AM said:

Hi Phage, I am officially a fan of yours

I just have a question regarding your post. I usually add little amount of ligation product to my transformation, but I do it because I was told so. Nobody has NEVER explained to me why adding 10 uL or more of ligate will result in a low efficiency transformation. Could you explain me that?

Thanks

As with many other things having to do with transformation, the root cause of this is obscure. Presumably the addition of low concentration solution to the high osmolarity transformation buffer results in change in the salt concentration of the solution suspending the cells. Given their fragile condition, this may be enough to kill a substantial number of them, or to upset the dynamics of the transformation reaction. Short answer -- I don't think anyone knows. I once tried to formulate a 2x concentrated version of transformation buffer which could be used to dilute ligation product to the same salt levels as the competent cells. I had mixed success in using it, probably because of bad control over the concentrations and the pragmatic difficulties of doing large numbers of experiments with many many variables.