Unlikely that restriction enzymes survive the recommended heat inactivation. You could test it easily by using heat inactivated enzymes to digest your plasmid. More likely it's the cloning procedure. Do you have an overhang on the PCR product after the enzyme recognition site? How long do you digest the PCR product? Typically I'll digest a plasmid for 1 h but a PCR product for 3-4 h.

Hi Nasir,

I think that Leila is right about inactivation. Double check the required flanking sequence for both enzymes and the activity of each enzyme in the double digest. You may need to go longer as Leila suggested.

Good luck,

Shale

Dear Leila and Shale,

That's a good way to check the efficiency of heat inactivated ones I will do that. Yes I have added a landing site (6nt) for both enzymes. I first optimized the digestion time on vector (1hr) and then did the same for PCR product. But thanks for your suggestion to increase the time for 3-4 hrs in case of PCR product.

In case my enzymes can't be heat inactivated then is there any way to get rid of their activity without affecting the DNA quantity much. Gel purification might be the best way but I am afraid of significant reduction in DNA. Any alternative please?

I just want to share with everyone that I did check the activity of heat inactivated (80C for 30min) restriction enzymes (AsiSI and NotI) on vector and it is negative. So the conditions mentioned above or on company website can be followed.

Hi Nasir, I think another way to remove enzymes is something like a phenol/chloroform extraction, but that would probably also be associated with loss of DNA (same as gel purification). Anyway it sounds like the heat inactivation is working, good news. I think increasing the digestion time on the PCR fragment will help. Another suggestion that you may have already considered: a common misconception is to add a lot of PCR product to the digestion. But this actually decreases the likelihood that you get a double-digested PCR product. Better is to add a controlled amount (~1 ug or less) so that both enzymes get a chance to digest the same fragment. The reason for increasing the incubation time above the 1 hour to 3-4 hours is that the ends of the fragment are moving around more so harder for the enzyme to bind (that's how I always thought about it anyway, might be nonsense). Same concept as adding PEG to a DNA ligation or ligating at 16 degrees C--- reduces the kinetic energy of the reaction components. Last point: try ligating overnight at 16 degrees C for maximum efficiency.

Hi Nasir,

All great suggestions regarding how to improve enzyme efficiency. You can readily remove enzymes with a standard QIAGEN PCR clean up, gel purification, or phenol/chloroform.

How big is your insert? You may want to increase the ratio of insert to plasmid. I generally go 10:1 with pretty good results. Alternatively, but not optimally, you can TA clone your amplicon, cut that, and use the insert for cloning. This will ensure that you have the correct ends and that the product is digested.

Good luck,

Shale

Dear Leila and Shale,

Thanks again for your valuable suggestions. Actually my total DNA amount of gel purified amplicon was around 1.7ug (48ul) so I put that all in 60ul double digestion reaction (by increasing the enzyme units accordingly) for 4 hours (kindly suggested by Leila) followed by heat inactivation (conditions mentioned above). Will it be ok? After digestion, I set up ligation at three vector to insert ratios (1:3, 1:5 and 1:10) last night by keeping the vector amount 100ng (16C incubation overnight) and will electrotransform today. Let's see the outcome. My insert size is 621bp and vector size is 6312bp. TA cloning is the best option but unfortunately I don't have kit for that. PEG is also good option but I also don't have that in lab. Any alternatives?

I think the conditions you used look fine. 16 degrees C for the ligation is great (don't need PEG). I agree with Shale increasing the insert:vector ratio is probably good considering the sizes in this case (the ratios you used look good). 1.7 ug of digestion substrate is a little high but not too high. It could potentially work! Do you plan to transform the whole ligation reaction? I recommend doing so. I also recommend doing the 1h incubation of the comp cells + DNA on ice prior to heat shock (or however you transform). Because you may have only a few ligation products, we want to maximize transformation efficiency. Let us know! Good luck!