ligation and transformation problems! Urgent! - (Nov/30/2004 )

I do a digest at 37 °C for 3 h, 10 min aT 65 °C... Perhaps it is better to do a sequential digestion.
But I make no difference in incubation time between vector and insert.

---

Hi

It has been some while since i made ligation and transformation, but have you calculated the ratio vector:insert in grams or in molar??? There is a big difference when they have such a great difference in size.
I think it should be in molar (=number og molecules)

Regards Caro

---

I try ligation with the ratio based on the molar ratio. I have ever tried the ration in grams. But neither works. If the ratio is in molar,only a very small amount of insert has to be added. If the vector is not completely double cut, is there much more chance for vector to self-circularize?

---

Hi guys,
I'm not sure about the amunt of DNA to do tarsformation because when I do trasformation to do for example maxiprep ( in any case not DNA from ligation ) I used 1 microgram of DNA and 50 ng of DNA to compare the efficiency.when I use 1 microgram the efficiency is more high than 50 ng.
I think the limit of amount of DNA is for ligation. Don't you think?
I'm sorry for my english.

---

HI ,
To standardise the ligation reaction one easiest step u can follow is
digest ur plasmid with one of ur enzyme and ligate back in diffrent conditions
and look for colonies .
perform a transformation with these contols
1) uncut plasmid
2) single cut plamid
3) single cut plamid + ligase
this approch at least will allow to check where is the problem??

bye
praveen

---

Thanks for your advice! I do try all the steps you say and the single cut plasmid+ligase grows a lot of colonies while single cut plasmid not. The problem is mostly likely the second digestion especially in this case BamH1.
I am going crazy in this step!!


netnus

---

HI,

So at least u come to know that BamH1 is not cutting ur DNA. Did u try the transforamtion with Plamsmid cut with only BamH1. If u observed colony in this step then something serious with plasmid preparation. Or u can do one thing
----First cut ur plasmid with BamH1 and then gel purified the fragment (linear one) to avoid uncut plasmid
----then cut with ur second enzyme. and set up a test ligation.
This step surely will give u some posotive results.
bbye

---

I don't think that it is necessary to purify by gel after first digestion. The loss of DNA could be to high... But it is necessary to refresh buffer after first digestion.

---

Things are getting better! I finally see some colonies in the plate. I pick all of them and grow in the LB mdium.
After extraction, I run PCR and yes,I can see the pcr product in the right position. Next I would try double digestion to see if I can see the insert band.
Thank you all very much for your generous helps!

netnus

---

And what did you change at least?

---