Is the problem that the enzymes are not digesting your DNA? Or the banding pattern does not match your expectations? Or that your insert and vector are the same size and do not easily resolve as two distinct bands?

How do you know your double digest is working? How do you know that an insert is present?

my insert is approx 1.6kb and vector is 4kb. i have checked the digest on gel with my undigested plasmid. so i can see linearization of the digested plasmid.

The last time I digested 10 clones out of which one seem to work. I am not sure where I am going wrong?

when I am expecting two separate bands for vector and insert, I can see one single band of around 6Kb.

The buffer requirements for these two enzymes is quite different, so perhaps your double digest is not working because the conditions do not allow both enzymes to be active. Alternatively, one of your enzymes may have gone bad. Are you using NEB enzymes? If so, what buffer conditions? Can you try sequential digestion instead?

Also, you might check your plasmid map again. Many TOPO vectors (at least the Invitrogen vectors) contain EcoRI site 5' and 3' to your insert. Single digestion with RI or another suitable enzyme may release your insert.

I agree with Daniel, you should check the buffer compatibility for the two REs. I am curious why did you do digestion for 3 hours, normally 30-45 minutes is sufficient to see cleavage. Such long incubation may give you some strange results due to star activity of the RE.

Apoorva- somehow overlooked the second part of your post. NotI has 25% activity in the CutSmart buffer, and HindIII, only 50%. This is not a good choice. Instead, digest in NEB buffer 2 for HindIII, then add NaCl (from 1M stock) to bring the final concentration to 100mM, supplement with NotI, and complete the sequential digest. 30-60 min for each digestion.

The high fidelity versions work for 100% in the Cutsmart buffer:

https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes

So the issue seems to be something else. Your previous experience (1/10) is not informative enough to tell you how many clones you should grow. You can screen through lost of colones using colony-PCR, and then grow the positive ones for plasmid isolation and verification.

Maybe these aren't high fidelity version of the enzymes?

Thank you all for the replies.

to start with

1)the enzymes are high fidelity enzymes, I forgot to mention them before

2) both the HF enzymes have 100% activity in cutsmart buffer

3) I have tried single digest with Hind III and observed linearization of Topo vector

4) also I have introduced the Hind III site in my PCR product at the 3' end and NotI site is present in the polylinker region of the vector.

5) I am trying to optimize my Topo cloning reaction as of now.

I suspect that my insert is in the opposite orientation . Is there anyway I can avoid wrong orientation of my insert during cloning. How Do i screen my colonies before stepping forward??

Which vector are you using? A TOPO-TA vector or a directional TOPO vector?

Daniel-After separating my insert from the Topo vector (invitrogen) , I need to ligate it into my another expression vector. So I have selected the restriction enzmyes in such a way that they are present in my expression vector for easy ligation thus limiting my options for selecting RE.

Topo TA vector (pCR4-topo)

You may have been unlucky enough to get the insert in the wrong orientation. Try screening more colonies to find one in the desired orientation. In the future, you might incorporate your 5' cloning site into your PCR oligo.

There is the slight possibility that there is a selection pressure for the inverse (wrong) orientation. This would require T7-mediated expression, and a toxic or deleterious effect on bacterial growth. This rarely happens, but is a potential consideration.

Unlucky- that is what my supervisor said.

Thank for the advice and suggestions Daniel.

I am screening more colonies tomorrow. Hope to get back here with some good news.

Cheers

For the issues pointed out above, I only use Fermentas enzymes (Fisher carries them) because they have 176+ enzymes that are 100% active in one buffer AND the digestion reaction typically only takes 5 minutes. They have a 12 pack that is only ~$225 and includes both of your enzymes. Big time saver.

Update, New England Biolabs will be transitioning by the end of May to a similar system as Fermentus, with 200 RE that cut in one buffer and in 5-15 min. Great to see, since I started out in grad school using NEB enzymes.

As for the orientation issue, try screening 10-20 colonies, if there is no toxicity issue then you should be able to find a insert in the right orientation. Also, have you taken a clone with insert and digested it "separately" with Hind III and Not I? If each RE linearized the plasmid by itself, but when used together do not drop out the insert, that would confirm that the insert is in the wrong orientation. So in the future, always put a RE site on the 3' and 5' primers and then you will not run into this problem again. You could always just order a new 5' primer with a RE in it and start over, some times that is the best way.

Let us know how it goes and good luck!

If I were going to do what you are doing, I would have just engineered the Hind III and Not I RE sites into the 5' and 3' PCR primers with enough extra nucleotides 5' of the RE sites in the PCR primers so that I could just "digest the PCR product" with Hind III (+3 bases past RE site) and Not I (+1 base base past RE site), use a PCR spin column (Sigma) to purify the digested PCR fragment away from the cut ends, and then clone the digested PCR product directly into the final vector that was cut with Hind III/Not I and "carefully" gel and spin column purified (Sigma). NEB has a chart showing how many nucleotides past a given RE site you need to get efficient cleavage by a given RE, see: https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments

While Hind II cleaves 50-100% with 3 bases past the RE site when using a FAM labeled oligo, Not I only cleaves 20-50% when close to the the end of a DNA (1-5 bases) when using a FAM labeled oligo, but if you click the above link and then open the older tests which used a plasmid as a template, Not I cleaves+98% close to the end of a DNA when using a linearized plasmid.

I have used the TA vector in the past and it is a fast way to clone a PCR product, and if you just want to sequence the insert then it is good. But if you need the PCR product in another vector, then you have to grow up colonies, screen them, purify a plasmid, RE digest the plasmid, gel isolate the insert, and then ligate the insert into another vector. That makes the TA strategy very slow in the end.

I haven't use TOPO vector. I read that it is a temporary vector for insertion of PCR product prior to the targeted vector (please correct me if I am wrong). I used pSCB for similar approach. I am a bit confused with the wrong direction issue. Even if you inserted the PCR product in a wrong direction (which I experienced very often, also with SUMO vector that relies on Adenine overhang), double digestion with REs will give you the insert because the RE cleavage site is (mostly) mirrored. HindIII cleaves at AAGCTT whilst NotI GCGGCCGC, thus the complementary strands is the same, right?

@Wangsa- the issue is that the NotI site is in the vector in this case, and its orientation relative to the insert is fixed. The HindIII site is found only at the 3' end of the insert.

@Daniel- I see, thanks.

Hello, Apoorva! I have the same issue with restriction enzymed from NEB. I use EcoRI and SpeI. They are both HF enzymes. I have tried sequential and double digest. But nothing works. Could you give some advice? Thank you! 

I would first make sure the DNA used in the digestion was high purity and free of residual alcohol. Next, have you tried a control digest of lambda DNA (or known  "good" plasmid DNA) with the same RE to make sure your RE and cutsmart buffer are okay? The NEB enzymes should be good, I also use ThermoFisher/Fermentus RE and they have always worked good too.

Dear Gary, 

 I am facing the same problem with NotI and Mcrbc. I was using takara . Although the McrBc digestion was optimized using  bsa and gtp.Would the same be needed for the Not I too.