ligation failed - (Jul/28/2008 )
I'm going to insert a 380bp gene fragment into a 5kb vector. Because the initial insert doesn't have compatible ends with the vector, I added the BamHI and EcoRI restriction sites by PCR using a plasmid containing this fragement then did the TOPO TA cloning. Then I digested both the TOPO vector containing insert and the 5kb vector by BamHI and EcoRI. The corresponding bands were cut from the gel and extracted by Qiagen kit. The vector and insert concentration are 50ng/uL and 5ng/uL, respectively. So far everything worked.
Then I set the vector amount to be 100ng and calculated the insert amount. I tried 10:1, 5:1, 3:1, 1:1, 1:3 and 1:5 insert/vector ratio and the ligation mixture was incubated at 16C overnight. Then I transformed 50uL competent DH5alpha with 1, 2, 5 and 10uL of the ligation mixture. I only got ONE colony from the 10:1 and I can't repeat it! There was no colony in the BamHI/EcoRI digested vector plate. The positive control plate was fine although I didn't calculate the transformation efficiency.
I inoculated 5ml LB with Amp with the colony and isolate the recombinant vector. I digested the vector with BamHI/EcoRI. It seemed the band close to 400bp was very wide. When the recombinant vector was digested by ScaI or SacI/SacII for 2 hr, there was a lot uncut. I set up an overnight digestion to see if it can make a difference.
Although I'm going to send the recombinant vector for sequencing, I still would like to get some hints from you guys.
The ligase is brand new so it should not be a problem. I also tried to heat inactivate the ligase before transformation but it didn't work either. I searched this forum and found damage caused by UV light might be a problem. Is the insert concentration a concern?
What's about a positiv controll for the ligation (any ligation which worked allready)?
Sure that you didn't lost the DNAs after the extraction?
Sure for the calculations?
You can try my program, if you like: