Ligation problem - 2 Questions (Sep/05/2008 )
1. I am using the Zero Blunt cloning kit. I used the control DNA with the kit, 800bp insert you PCR. The PCR product was run on a gel and an 800bp band was apparent. I used the Zero Blunt vector and T4 ligase 16C for 16hrs. Product transformed with TOP10 cells and plated on Kan plates. No white colonies, only blue. Could it be that my Taq is added an A overhang and thus isn't blunt ended so is not ligating into the vector? HOW CAN I REMOVED THE A OVERHANGS? WHAT KIND OF TAQ OR OTHER ENZYME DOES NOT ADD A OVERHANGS?
2. Ligated product from above transformed with TOP10 cells and spread on Kan 50 ug/mL plates. After 16 hours, very small, 1/3 blue and 2/3 white colonies appeared. I waited 24 hours and came back and all colonies turned blue. Why? Did they lose plasmid? Or, all colonies contained no insert?
Most of the proofreading enzymes, such as Pfu and Phusion do not add A's. Any Taq or Taq/Pfu mixture will add A's. The descriptions of the enyzmes will usually include this information (except for Taq where "everyone knows."
The blue colonies probably do not contain inserts. You can make the blue more visible by putting the plates into the refrigerator for a few hours after colonies are visible. S-Gal (Sigma) will provide a more visible difference between white and blue (black for S-Gal) colonies.
i agree with phage434, i dont think the colonies lost the inserts. Blunt ends are too dificult to ligated
So how can I fix this? I don't think I can order another enzyme for PCR because of high costs most likely. What about repairing with klenow or T4 Pol?
I am in need of help. i am doing ligation of a vector(wt 515) in PGL3. but i am not getting my required result. i am doing as follows.
1. i design primer than do PCR with these primers got my reguired band in Gel.
2. Than i do TOPO and again got required band on gel.
3.than i extract this band using Qiagen gel extraction kit and digest with KpnI.
4. i also digest PGL3 with KpnI.
5. now i am doing ligation using T4 DNA ligase but every time i got contaminated colonies. means when i extract DNA from these colonies using Mini Prep and than again cut with KpnI enzyme i got nothing. positively i should got 2 bands one for PGL3 and other of my vector.
I am trying this since last 3 months but m not getting plz give me some suggestions.
Never used TOPO cloning, so this might be entirely wrong...
Try CIP treating the cut vector. That way you'll not get any significant background.