I only have two possible restriction sites for cloning an insert into my vector. One end is a sticky end (bglII) and the other end is a blunt end (HPA1or KSPA1). I have never done a blunt end ligation. Can I ligate my insert in a single reaction with these two different type of restriction sites? Are the reaction conditions for sticky and blunt ends ligation compatible for single reaction? Has anyone done this before? Or would this combination make my insert incompatible using these two restriction sites and my vector size is 6.4 kb and insert is 2.4 kb so what are precautions has to take in transformation
Ligation conditions are different for sticky and blunt ligation. Since you are forced to do this I would simply try with conditions for blunt ligation. This might produce some insert dimers, so you might want to increase insert concentration. Given the size of the insert you can easily distinguish between the correct clones and some artifacts.
I would also suggest that you "fill in" the sticky end. This will tend to make the blunt end ligation more efficient. As BglII give you a 3' overhang, the Klenow fragment would be the obvious tool to complete this filling in.
Andreas is correct - as you have a quite large insert, it shouldn't be too hard to screen for the correct clones.
Hi there,
The alternative to Andreas suggestion (increasing insert concentration) is to increase T4 ligase concentration according ThermoFiher/Fermentas protocol.
It´s not necessary to fill the sticky end and we have never seen dimer insertion in our cloning attempts; this can only happen if your vector has been cut be one enzyme and the insert dimerizes first, which might occur with high concentrations of insert.
I have never made differences in the ligation conditions when one end was blunt and the other sticky. Typically I ligate at 14C overnight, but 4hrs at RT also works, with 1µL ligase.
Agree with Eric, I've never tried ligation more than one step. I usually use a reaction system of 20uL with 1uL T4 ligase. Based on your insert and vector size, I would try different I:V ratios (i.e 1:1, 3:1, 5:1), and since you have blunt end, I would do a ligation at 16 degree overnight.
Ligation for blunt end takes more time than sticky ends due to presence and abscence of overhangs, to minimize this increase enzyme concentration. Blunt-end ligation is also reversibly inhibited by high concentration of ATP. Prefer to keep blunt end ligation conditions. There is chance to form unwanted ligations if we kept with sticky end conditions. I suggest you to consider all the suggestions and plan yourself
dear all the above suggestions are equally good. you digest you vector and insert DNA and purify them using column separately and set a ligation reaction with vector & insert molar ratio 1:6 or1:5. now set the reaction at 8 deg C for overnight (it works best for me for all kind of ligation reactions ) since your insert size is large. it will increase the efficiency of the reaction as one of the ends is blunt, which needs lower temperature for ligation.
Thank you to all for giving valuable suggestions. Dear Aditya, do you use the PEG for the sticky and blunt end ligations? and how can we maintain inserts with out ligating to each other at their blunt ends in the ligation reaction (as Dr.Andreas said 2.4kb is ok. but if insert is
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