I want to clone my PCR insert into a pET-28 vector (which was isolated from BL21 DE3 using commercial mini plasmid kit) between BamHI and XhoI sites. For 4 hours I digested vector and insert in two different tubes (25 ul of total volume, 5 ul Tango buffer (x2), 2 ug of insert, 2 ug of vector, 0,5 ul BamHI (5U), 0,5 ul XhoI (5U). Then I ran 3 samples on 1,5% agarose gel with EtBr:
1-double digested PCR insert,
2-double digested vector,
3-undigested vector (0,5 ug).
As a result I see one band of 1 (correct), two bands of 3 (correct) and there were no bands of double digested vector at all, completely disappeared. Why is that? Does anyone have an idea what could have gone wrong? I repeated the digestion twice, unfortunately with the same result.
There are some extra information which would help to understand better the problem:
1.- At which temperature do you do the digestion? I supposed it was at 37C, but just to be sure.
2.- How many volume of the digestion reaction (1st and 2nd samples) are you loading on the gel?
3.- You said that the 3rd sample (Undigested vector) show two bands and was correct. I couldnt understand why you see two bands, please could you explain at what size and why did you expect those bands?
4.- How do you confirmed that you obtained the plasmid after the miniprep?
For example: In my experience, After the miniprep is done I digest the vector with 1 enzyme to linearize it, Load it in to the gel and expect a Band next to the size of the vector.
Probably, I might not have a solution for your problem but I am sure this information could help to get that solution.
Best,
I agree with Jaime, it's baffling that you expect to see 2 bands from the undigested vector!? What's the vector size? While it's quite unlikely, at least I haven't heard of it, could it be that your enzyme is contaminated with nonspecific restrictases munching through everything! May be shorter restriction (2 hours) time will clarify some of the issues
Some answers to your post Jaime:
1 - 37 degrees
2 - I'm loading everything (25 ul of sample + 5 ul of loading buffer), then I'm cutting the gel and isolate DNA with a kit
3 - Two bands correspond to supercoiled and nicked circles forms of vector
4 - Well I didn't, I only check the purity running the isolated vector on agarose gel
Vlady, the vector size is about 5000 bp. I guess, maybe the plasmid is somehow contaminated?
I'll try to isolate it once more and then do single digestion with both enzymes separately, maybe it will work, thanks for response!
Artur,
It seems to me that you would have to perform a digestion with an enzyme with two sites first (most vectors have EcoRI) , and see if you have the insert cloned in. Or, alternative, go to a rapid PCR. In both cases you will see a fragment with the expected size. You can also try sequential single digestion.
I agree with Walter Papsergi respect to DNAse contamination. Take a little aliquot of you vector and incubate it at 37°C (or the temperature that you use to incubate with your restriction enzymes). If the vector desappear then is a contamination.
But if there is DNAse contamination, why the undigested vector do not "disappear". It has to do with something in the digestion buffers...
Have you tryed the same reaction with a new buffer? Is it possible for you to try a different set of buffers?
I would recommend to digest 1-2 hours with 5 units of each enzyme, but remember to add BSA! Both BamHI and XhoI needs BSA to avoid star activity! I don't believe in contamination. You have most likely used the same buffers and enzymes for the digestion of the PCR insert, right? If that does not degrade, you do not have a contamination. You ran your undigested vector on the gel, so you know the miniprep worked and can see the approximate concentration.
I think you should do a plasmid miniprep from different E. coli strain (in my lab DH5alpha works well, JM109 and XL-1 Blue will be fine as well). BL21 is for protein expression rather than plasmid preps I think it contains an additional plasmid (pLys).
-I recommend to do at least 2 miniprep of different colonies, in this way it is easier to equilibrate during centrifugation, and you have better chances to obtained what you want.
-Always perform a confirmatory of the prescence of your plasmid, it can be done by amplifying a fragment with PCR or Digesting with one enzyme and expect the size of the plasmid. In my personal opinion, I wouldnt use the undigested vector as a confirmation because it is supercoiled and can present differents structures that led to multiple bands on the gel that can be misunderstood with RNA(in the case you are not using RNAse in the miniprep) or Fragmented Genome DNA contamination.
-Even though 25 ml for the reaction mix can work well, its always better to work with the maximun practical volume (I usually used 50ul), in order to dilute any contamination that could inhibit the reaction.
-As Sine Topp said, both enzyme need BSA in the reaction, but if I am not wrong you are using Fermentas Restriction Enzymes with Tango buffer for double digestion (Yellow), this buffer already contain BSA so it should work fine. If you are not using that buffer make sure to have BSA in the reaction mix.
I hope you can solve your problem, if i another idea come to my mind i will write it inmediatly.
DNAase contamination from de BL21 strain... check that is EndA1-, SOLUTION: purifiy your plasmid with a gel purificacion kit, use it as the volume of plasmid is agarose.... repeat the digestion....good luck
.Perform the double digestion in two step and control on agarose gel each time . Check for the reaction buffer or other (water..)adding only one reagent to the reaction and after incubation at 37 °C control on agarose gel.
Good luck
Problem would be with Vector, It might not be purified DNA. DNase contamination would be present. Check for Nuclease Activity by keeping 500ng - 1microgram of Vector DNA at 37C or RT. Other way, dont do double digest, do it separately and check the Enzyme contamination also.. All the best.
Hey! Thank you all for helpful advices! Fortunately the problem has been solved. It was DNase contamination of plasmid. Today I used other kit for isolation, and this time I tried precipitation with ethanol. Last time for the first time I used AxyPrep from Axygen and I skipped additional washing step for endA+ strain. A kit I usually use has got only one washing buffer and it works fine, thats why I skipped additional using AxyPrep. I checked and BL21 DE3 is endA+, so here we have explanation. After digestion of freshly isolated vector everything works fine after 2 hours. As it turned out, the old vector after incubation at 37 degrees without restriction enzymes was totally degraded. Thanks once more :)
Today, I am facing with this problem when performing double digested vector. Thanks a lot for your information.
I have the same problem where after performing double digestion I see no bands at all in the gel. I don't see a band for the digested DNA fragment as well. and i am not sure that a vector would be degraded if incubated.
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