Hello everyone I'm trying to pseudotype an oncolytic virus by restriction enzyme cloning. I have generated my insert by PCR by adding the necessary cutsites. I only have one restriction enzyme site I can use in my vector ( in this case, the virus I want to pseudotype) so directional cloning is not possible.
I first PCR purify my insert then cut both my insert and vector with the same enzyme at 37C for 2 hours. After that I use quick CIP from NEB to dephosphorylate my vector. I heat inactivate the enzymes and then move on to overnight ligation at 16C with T4 ligase.
I transform vector only, vector and ligase, vector ligase and insert. I then screen my colonies by colony pcr for the insert. Even though I've screened more than 20 colonies, I still haven't gotten a positive hit for my insert.
My background in vector and vector-ligase transformations is on the high side. So I thought that the vector was religating with itself (as the enzyme I use generates sticky ends) and increased the incubation time with quick CIP to 1 hour. It did reduce the background a bit, but it still looks like I keep getting empty vector. I have tried two different ratios for ligation (3:1 and 5:1) but that still has not changed the result.
I really don't know what the problem is and I'd appreciate any help. Thanks!
Dear Bengisu
did you purified from gel your digested vector or did you performed standard coloumn purification kit?
Because your problem can be associated to the presence of some un digested vector that is able to trasform e.coli and provide the background.
Consider that for example is you use 20ng of plasmid for the ligation in case you have 1% of non digested whixh are 0.2ng with good competent cells (competenze >10^6 cfu/ug) this may result in more that 100 colonies.
However for the future, evaluate the possibility to use an enzime free cloning approach as PIPE cloning
you can find detail about it on my blog;
ProteoCool (https://proteocool.blogspot.com)
ProteoCool n°1 (Cloning methods overwiev) and
ProteoCool n°3 (Primer Design: PIPE Cloning
at page 1.
good luck
Manuele
Dear Bengisu
did you purified from gel your digested vector or did you performed standard coloumn purification kit?
Because your problem can be associated to the presence of some un digested vector that is able to trasform e.coli and provide the background.
Consider that for example is you use 20ng of plasmid for the ligation in case you have 1% of non digested whixh are 0.2ng with good competent cells (competenze >10^6 cfu/ug) this may result in more that 100 colonies.
However for the future, evaluate the possibility to use an enzime free cloning approach as PIPE cloning
you can find detail about it on my blog;
ProteoCool (https://proteocool.blogspot.com)
ProteoCool n°1 (Cloning methods overwiev) and
ProteoCool n°3 (Primer Design: PIPE Cloning
at page 1.
good luck
Manuele
Manuele Martinelli, thank you for your answer! I have not gel purified or PCR purified my vector after digestion, I immediately moved on to the ligation step. However, I have run both my digested vector and digested PCR product on a gel to see what they look like. Because I am only using one restriction enzyme, it is not possible for me to check if my vector is digested and isolate the digested part from the gel via gel purification because I only get a single band. Do you think extending the restriction enzyme digestion incubation time would help ensure that all of my vector is digested (maybe even overnight digestion?)
I will also definitely check out PIPE, thanks again.
Dear Bengsiu
generally closed vectors differed from the digested because it show on gel more conformations, as coiled, supercoiled, that do not happen for the digested. Therefore in your place after incubation with cip and i will try to perform gel purification of thr digested vector and use it for the ligation also if you have a single digestion.
Of course a single digestion increase also che probability of re-ligation therefore it is not certanilly a simple cloning.
Actual dìgestion enzime are quite efficient, so you can try to increase the time but i'm not sure that it will change a lot.
with your actual experiment desing the only things that i can suggest to you is:
- purify the digested vector from gel
- increase the ratio insert/ vector (1:10)
- screen many colonies
because restriction enzime cloning with a single enzime is not a simple approach.
good luck
Manuele
Hi, after ligation, run the samples in an agarose gel electrophoresis and check the size of the ligated product (the vector + the insert) in comparison to the vector alone.
Can you give here what is the restriction enzyme that you are using and the complete protocol?
You may refer to my following publication, in which we have used Bam H1 as a simple cloning to insert the cDNA of one of the adjuvants (cytokines) in the expression vector, pcDNA3 plasmid.
Article Enhanced protective response and immuno-adjuvant effects of ...
somasundaram C et al 1999
Dear Bengsiu,
Extending the incubation time leads to non-specific digestion of the insert known as star activity. So keeping the minimum time of digestion and purifying the insert and vector directly, rather than from gel, check the concentration by measuring the intensity of the band. Let say, load 2ul of the sample of each.
Then setup a ligation reaction with vector:insert in 1:3 ratio. Hope you can troubleshoot this problem. Good Luck.
Chandra Somasundaram thank you for your reply. I will definitely refer to your article. When I ran my ligated vector, it did not show up on the gel, I read that that may be because there is very samll amount of ligation, but I will try again.
I use Acc651 an isoschizomer of Kpn1. It leaves sticky ends and can be heat inactivated.
For the restriction my protocol is:
acc651: 1 microlitre
DNA: 1 microgram (for both the vector and insert)
3.1 Buffer (suggested by NEB): 5 microlitre
dH2O: up to 50 microlitre
I then incubate at 37C for 2 hours. At the end of the 2 hours I add 1 microlitre of NEB Quick CIP, incubate for another half an hour, and then inactivate all my enzymes at 80C for 20 minutes. I add CIP only to my vector and not my insert.
For ligation:
I use 5:1 and 3:1
Reaction buffer: 2 microlitre
Vector DNA: 100 ng, insert calculated accordingly
t$ ligase: 1 microlitre
dH2o: up to 20 microlitre
I have tried 10 min incubation in room temperature and 16C overnight incubation, followed by 65C 10 min heat inactivation in both cases. After this, I transform vector, vector and ligase, vector, ligase and insert to e.coli.
If you have any suggestions regarding my protocol, I would greatly appreciate it. Thank you!
If I were you, I do the following
1. Gel purify the digested vector (and the insert) after treating with CIP to avoid background due to uncut vector (run uncut vector in a separate lane as a marker to make sure the digested vector separated from the uncut vector) and to remove CIP, which can dephosphorylate the insert in the ligation reaction if it is not completely inactivated/removed. If you are still unsuccessful, try point two.
2. If possible, use two different restriction endonucleases that produce compatible cohesive ends to digest vector and insert (For example, digesting vector with BamHI and the insert with BglII), treat with CIF and gel purify. After the ligation, digest the ligation mixture with the restriction endonclease specific for the vector (which would have lost in the recombinant vector that has the insert) so that you can get rid of any residual uncut vector co-migrated with the linearised vector. This means you have to make new primers. If you are unsuccessful with the cloning by this way, please try the point 3.
3. Try ligation free cloning using the kits available from NEB or Clontech.
Venkateswarlu Kanamarlapudi , Pratibha Srivastava
Thank you so much for advice, I will try them out!
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