Ligation of 2 fragments and one vector - (Jun/15/2005 )

hi ,guys

I've tried to ligase 2 fragments and one vector(pcDNA3.1+) together at the same time.But I didn't get the cloning at last

The two inserts:

"A" (400bp) is with Hind3 and ApalI sites,

"B" (1.7Kb)is with ApalI and XhoI sites, and the vector is with Hind3 and XhoI sites

vector(~5.4kb)

Methods:

I 've tried

1) Ligase A and B first and then insert them into the vector

2) Ligase 2 fragments and the vector together at one time

The ligation I usually took was at room tempreture for 16hrs

And then set the DNA transformation, sometimes I could not get any colonies.

If I got the colony, and it is still not the cloning I supposed.

I am confused about the results, I have checked all the fragments, and extracted them separately!!

Could I have your helps please ? and what do you think about the process?? Is there any improvement and possible mistakes....

Thanks a lot...

Looking forward to your replies

sincerelly

---

Hi
I am not sure exactly but possible reason that strikes my brain is it could be with the Ligase.I usually use PROMEGA T4 DNA ligase and i do the ligation reactions at not more than 10 microlitres and at 4 C overnight.The product catalogue also says 15 C for 4-6 hrs.

Hope that will work.

Thanks and Good luck

-S

---

Hi, do you set the control for ligation and transformation? Try to set one control for ligation and one control (uncut plasmid DNA ) for transformation. Based on your description, there are probably problems with the ligation. I am using the pGEM-T vector and the ligation condtion is 4 C, overnight or 15-16 C 4-6 hrs. There is no problem with my ligation.

Besides, if you get the fragment from gel, there are probably problems with you DNA. Hope this will help.

---

Thanks

I use the T4 as well...and mine ligation is 20ul...I don't know if the volumn will affect this reaction.......

Anyway, I will try and have a look.

Btw, what's the ratio did you set of the vector and insert fragments??

---

Thanks a lot. Good point^^

Yes, I 've set the control of ligation,but it is the vector with two enzymes sites not the uncut plasmid.

I don't think my DNA is not ok. So have you met any problems with the LB+Amp plates???If the Amp is too old, maybe we could not get any colonies...is that right???

Anyway, I will have a go of your suggestion.

---

Hi,
it may be a good idea that you run the reaction mixture before and after ligation on agarose gel side by side.
If the two samples look similar, ligation reaction may not be OK.
If LB+Amp plate is too old, you usually have too many colonies.
But the plate becomes dry, maybe you don't have any colonies.

---

Right!!! Thanks...

I don't know why my plates are so dry (btw,I have pre-warmed them in the incubator for 1hr before transforming).......you know, when I made the plates,it became dry so soon...and i could only spread them for 2 or 3 secs.
So I didn't get any colonies.

I've run the reaction mixture of the ligation of the two insert fragments. But there is not only one fragment as I supposed to be. There were also some bands whose sizes were double "A"size and double "B"size. (do you think they can ligase by themselves)

It made me confused if the ligation of the two fragments worked.or if the efficiency of self-ligation is higher or lower than the between-ligation??..

Have you ever met this thing before??

Thanks

---

Hi,

You saw many bands after ligation, which means your ligase is working.
However, it is difficult to know exactly where you see the correct product, because this is circular DNA.

I just recommend that you include positive control, especially uncut plasmid in this case, as Beaver said.
This will make sure that your competent cells and plating techniques are ok.

Good luck!

---