using diluted ligase - (Jul/31/2006 )
I have diluted T4 DNA ligase 1:50 x so taht my final conc. is 8U/microliter. I tried using 1 microliter in a 10 microliter reaction adn it didn't work, no colonies. DId anyone of you guys ever tried dluting T4 ligase and it worked?
we are using 3 units of T4 ligase to ligate 50ng vector + insert for a 20microliter reaction. (our stock is 3u/microliter). are you sure your T4 DNA ligase stock is 400u/ul. that's pretty much i guess.
i also wonder how did you dilute your enzyme? with water? enzymes have different storage buffers that they survive in until the expiration date. if you diluted your ligase with some buffer in which T4 will not be happy, then i doubt it will work properly
I think you've got to make sure that you've got the unit right. Just want to mention that there are 2 different units commonly used in the market: 1. Weiss unit 2. Cohesive End Ligation Unit. Each unit actually refer to different amount.
It appeared to me that you may be using T4 DNA ligase from NEB, which uses Cohesive End Ligation Unit (=0.015 Weiss unit). If so, the concentration of your stock T4 DNA ligase is merely 6 Weiss units. If you diluted it 50 X....I think you knew where I'm getting to. I normally use 3-6 Weiss Units in a 10 uL ligation volume. That would be 0.5-1 uL of T4 DNA Ligase from NEB, undiluted, labeled 400U (Cohesive End Ligation Unit)/uL.
This may sound obvious, but I have eyewitnessed two members in the lab made this mistake.
Hope this help. Wish you all the best in your research.
Thanks for the answer and you are absolutely correct! I have bought a ligase from Fermentas and I was surprised to see that they are insisting on writing on the tube that the concentration is 5 Weiss units so I checked it up on the net. So I guess NEB is the only company that states enzyme concentration in cohesive end units and I think my mentor didn't know that and was really surprised that they are even selling something so concentrated. Well, thanks anyway, I will share my wisdom with my colleagues tomorrow;)!