17 replies to this topic

[Ayukawa]

Hi all.
I've been struggling with one transformation for months and I really need some advices from you guys.
This is my case:
I have an 1.7kb insert and it has been double digested w/ EcoRV and XhoI to create a blunt end and a sticky end. I performed the same (double) digestion on the vector (pcDNA3.0-FLAG, 5.4kb).
Next, I performed the ligation (so far I'd tried 2 ligation kits: Takara DNA Ligation Kit & Fermentas Rapid DNA Ligation Kit) and then I immediately made the transformation into DH5a cells.
However, I can rarely get colonies w/o insert using Takara Ligation kit and no colonies at all using Fermentas DNA Ligation kit. I have no idea what had gone wrong since I followed the protocol accordingly.
I'll write down the steps which I'd done during the ligation and transformation. Please comment.  

Ligation - Takara kit
Different ratio of insert:vector volumes (from 10:1 to 3:1) had been tried.
Same volume of ligation buffer was added and mixed

The mixture was incubated @ 16degC for 30min
10ul of ligation mixture was used for transformation

Ligation - Fermentas Kit
3:1 of Insert:vector (volume ratio) had been tried so far
4ul of 5x ligation buffer was added
1ul of T4 DNA ligase was added
Autoclaved H2O was added to fill the ligation mixture up to 20ul

The mixture was mixed and incubated @ 22degC for 5min
5ul of ligation mixture was used for transformation

Transformation (DH5a)
Each tube contained 50ul of competent cells was used.
The cells were thawed on ice for 10min after taking out from -80degC
The ligation mixture was added directly into the tube
The tube was rested on ice for 30min
The cells were heatshocked @ 42degC for 20sec
The tube was rested on ice for 2min
950ul of prewarmed LB was added to the tube thereafter
The cells were incubated @ 37degC waterbath, shaking @ 300rpm for 1h
200ul of the mixture was spreaded to LB plate containing ampicillin (twice)

TIA.

[pesji]

Ligation of Blunt end DNA is fa less efficient than sticky ends   Promega use to have a great ligation buffer which contains PEG and was in my hand working very well with Blunt ends. Be sure to lower the temperature of the ligation due to the movement of molecules are higher at temperature above 4°C. Maybe I would try a ligation overnight at 4°C with a buffer containing PEG.

[little mouse]

I always perform blunt end ligation at 16°C over-night whatever ligase I use.

[Clare]

little mouse, on Aug 4 2009, 09:01 AM, said:

I always perform blunt end ligation at 16°C over-night whatever ligase I use.

I agree - I always do a 16degC ligation overnight for blunt ends. You can also add 2ul of 50% PEG 4000 (for a 20ul ligation) to increase the chances of the blunt ends ligating (it worked for me in my magic 3 way ligation with blunt ends!).
Good luck
Clare

[lizzy]

I also do a ligation at 16°C overnigt and the result is very good.  In transformation , I think shocking the cells  at 42°C water bath for exactly 90 seconds is much better.

[almost a doctor]

Agree with everyone on the overnigh ligation.  You can always transform part of your mixture after ligating for short time, and leave the rest overnight to transform the next day if no colonies observed.

On the transformation:  do you have a positive control for transformation?  I think 10ul is too much DNA for the cells, and definitely heat shock at 42C for at least 60sec (I also always do 90sec). Also, I usually only add ~250ul after the heat shock and 2min on ice before incubation at 37C. Alternatively you could spin down after the 37C  incubation to concentrate the cells before you plate.

[Ayukawa]

Thanks for all the prompt comments and advices.  
I'll try overnight ligation and let you all know about the outcome.

lizzy & almost a doctor:
I'll try to extend the heatshock period to 60 sec; I hope I won't cause any harm to the cells

In fact, the amount of LB added prior to 1h shaking @ 37C was also one of my concern since I'll have about 600ml of them left after spread plate. I'd like to find out any good recommendation to break the pellet after spinning them down? Gently tapping on tube wall or...?

Thanks for your advices  

Best,
Ayu

[little mouse]

I agree with the amount of ligation product for transformation.
I use no more than 2 µL for 50 µL of bacteria.

For the plating, what I do is :

I add LB too, the final volume is 1 mL.
I plate 100 µL, and I centrifuge the 900 µL left, resuspend in 100 µL and plate it too. Then I get  10 times more colonies on this second plate.

[Ayukawa]

Dear all,

I failed my transformation, again. No colony found on plate1 while satelites were spotted on plate2 (which I think could be due to contamination).

I run a gel on the ligation mixtures which I left on last two attempts (5 min ligation L3 & overnight L4), a ligation w/o insert (control insert-) L5 and the insert + vector (control ligase-) L6 (please ignore L2). Kindly refer to the attached gel image and comment.

Thank you.

BR,
Ayu

Ligation

Edited by Ayukawa, 06 August 2009 - 08:12 PM.

[little mouse]

I know it's a little stupid question, but it has happened here right now : could you check again that you are using the right antibiotic?
second, could you have a control where you cut your vector and re-ligate to be sure your ligase is working ?

[Ayukawa]

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

I know it's a little stupid question, but it has happened here right now : could you check again that you are using the right antibiotic?
second, could you have a control where you cut your vector and re-ligate to be sure your ligase is working ?

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.

[virusfan]

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

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

I know it's a little stupid question, but it has happened here right now : could you check again that you are using the right antibiotic?
second, could you have a control where you cut your vector and re-ligate to be sure your ligase is working ?

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.

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.

[phage434]

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.

[Ayukawa]

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

Edited by Ayukawa, 17 August 2009 - 11:38 PM.

[memo]

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