details: vector and insert are 2.8 Kb, both spin coloumn purified and REN checked.
REN sites were introduced into insert (with 5bp extra sequences at 5`end).
O/N REN digestion followed by CIP treatment of vector . tried ligation with both quick ligase and conventional ligase.
colony PCR of transformants does shows up with some positive clones but plasmid prep shows it to be negative.
Hi Ramya, are the primers you used for colony PCR specific to your insert and have overlapping sequences with the vector? That may improve the selection process by colony PCR. Did you set up a positive and a negative control?
I use a lot of inserts (1:10) when I'm doing ligation. But now, our lab prefers to use Gateway recombination. It's really efficient! (:
After the ligation reaction you can check if the ligase was able to ligate your DNA fragments. Here you can find information on how this is done:
http://www.thermoscientificbio.com/whats-new-t4-dna-ligase/
If this experiment shows you that you have a positive ligation reaction then you can rule out the possibility that your ligase failed. It might at least help you coming closer to solving your problem...
Good luck!
In our lab, we switched from digesting the PCR products directly to routinely do subcloning in a TA-vector first and digest that one to get the insert which for sure hast been digested (you can never be 100% sure when digesting PCR).Good luck,Cindy
The temperature at which ligation is carried out could matter, you have to state at what temperature is your ligation reaction been done ? Your question did not mention at what temperature your reaction was set up , yes vector: insert ratio whether sticky or blunt ends has different mathematical equations that gives a clue
I completely agree with Cindy. You can digest your vector and easily check the efficiency of the digestion running the vector on agarose gel but you can not check if your fragment's ends are correctly digested and ready for ligation. Subcloning your fragment into pCR-TOPO vector allow you to digest the TOPO vector and ensure the correct digestion of both final vector and insert running both the digestion products on gel.
In this way you'll not have any kind of trouble. The time you spend on subcloning with TOPO TA (about 3-4 days) is well spent.
hi ramya,
If your transformants are positive by PCR, then u need not bother about ur ligation mix.
Obviously transformants will not be PCR positive if the vector with insert does not enter the host.
apparantly, its the plasmid extraction problem.try conventional plasmid extraction.
All the best
You could also have positive colony screening PCR without really having positive clones, it's not impossible. Bear in mind when you're plating the heat shock mixture which contain also the ligation mixture you'll have the desired fragment (and also eventually the vector efficiently ligated together with your fragment) both plated on LB-agar independently on the uptake of bacterias. If you're performing the colony screening directly from the plate and you're picking up a small amount of LB-agar you could have positive PCR from colony screening without positive bacterias.
I can also suggest you to test your bugs efficiency by plating also a positive control of transformation (i.e. pUC19 transformed bugs)
Perhaps the concentration of insert to vector ratio should be concerned.
Usually 1:3 to 1:5 is the perfect range. If you're in this range, you've verified the correct double digestion by subcloning and cleave the insert from a vector then everything should work (hopefully). What I can also suggest is try to perform the ligation overnight at 12 to 15 Celsius degrees in a cycler and finally disrupt ligase at 70 for 10-15 min. If even in this way your clones will be negative after the prep you should find the cause in the insert sequence (insert folding which cause interference during ligation or even sequences that are making your fragment prone to recombination or deletion in bugs).
When I'm cloning direct from PCR I use an excess of insert (1:10 - 1:30) in ligation. Check that you do not have match any contaminant in their ligation reaction . Ideally, your insert and vector are purified before ligation.
Check the integrity and quality of your PCR fragment by gel analysis. Optimize the insert: vector ratio as Lais mentioned above, try to increase insert concentration, set up different three ways of concentration 1:3 or 1:5 or 1:10. Also you should consider the time of incubation, ideally at 4°C (16-18 hours) overnight, when you use T4 DNA Ligase.
Hi Ramya, are the primers you used for colony PCR specific to your insert and have overlapping sequences with the vector? That may improve the selection process by colony PCR. Did you set up a positive and a negative control?
I use a lot of inserts (1:10) when I'm doing ligation. But now, our lab prefers to use Gateway recombination. It's really efficient! (:
Yes, it is very important the molar ratio vector:insert (1:5 is OK). Try with conventional ligation 16 hours at 16ºC and you do not forget heat the reaction at 65 ºC 10 min after O/N ligation.
Optimize the insert: vector ratio.Try 1:2,1:3 and 1:5.Make sure the enzymes you use are properly stored.
If RE directional cloning method is still not working, you can try the Gibson method (https://www.neb.com/products/e2611-gibson-assembly-master-mix). We use it routinely in our lab for cloning.
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