19 replies to this topic

[ranvi]

I have problem with my ligation, I cut my Baculovirus vector pAB  6xHis(9.6kb) with BglII,   BglII and XbalI since i have two inserts.
I  phosphatase the BglII cut vector. The same digestion i did with my insert (3.8kb) and gel purified it ( XbaI and BglII double digestion is 100:75 in buffer2 as perNEB) I did ligation with 1:2, 1:10  vec: ins ratio nothing works

Thank you for the help

[pDNA]

did a positiive control with a plasmid of choice to check if your competent cells are working? do you use electroporation or chemically competent cells?

Please give as much info as possible ...this will make it more easier to help you!

Regards,
p

[ranvi]

pDNA, on Jun 18 2010, 12:32 PM, said:

did a positiive control with a plasmid of choice to check if your competent cells are working? do you use electroporation or chemically competent cells?

Please give as much info as possible ...this will make it more easier to help you!

Regards,
p

[ranvi]

The cells i am using are  from Invitrogen CCDB oneshot cells which are specific for toxic gene ( I am trying to swap this toxic gene into baculovirus).
I am pretty sure the cells are ok. They have never been thawed out. I did work with that recently they are working fine. Did a transformation with a vector plsmid alone they work great.  gave plenty of colonies

The insert i am trying subclone is a toxic gene cut from a destination vector I made for gateway cloning( mamallian system). we are trying to do the same with Baculovirus vector so we could do it in a baculovirus system.

[pDNA]

so you cloning your toxic gene in your vector's MCS ...this step inactivates the ccdB gene and allows cell growth (i'm not that familiar with the gateway system ...so please correct me if i am wrong!). If so this is more or less a problem since upstream of the MCS there will be a constituitive promoter ...that will later drive the expression of your toxic gene and thereby prevents cell growth! Would this be possible?

Regards,
p

[ranvi]

pDNA, on Jun 18 2010, 02:45 PM, said:

so you cloning your toxic gene in your vector's MCS ...this step inactivates the ccdB gene and allows cell growth (i'm not that familiar with the gateway system ...so please correct me if i am wrong!). If so this is more or less a problem since upstream of the MCS there will be a constituitive promoter ...that will later drive the expression of your toxic gene and thereby prevents cell growth! Would this be possible?

Regards,
p

Although i am new to this my understanidng is  : The Toxic gene once inserted into baculovirus vector  it  will be destination vector and it is easy to swap the toxic gene with your gene of interest ie insert that you are actually going to work for expression, toxic gene is a cassette which can be swapped in gate way cloning

[pDNA]

can you provide the exact name of the vector u r using or can you provide a vector map?

normally if you do not get any colonies something goes completely wrong ...normally the problems is that you get to much background or stuff like this.

Therefore i would suggest that your toxic gene is too toxic for a high copy vector and the leaky promoter in front of it ....but this is only a hypothesis since i dont know the exact features of your vector.

Regards,
p

[ranvi]

Hi P,
Look for AB vector on google

Theproduct is

  pAB-6xHis™  Cat. #B4
  



DNA Sequence  
  
  pAB-6xHis™ plasmid transfer vector is designed for expression of proteins with N-terminal polyhistidine tag. A PCR fragment encoding a protein of interest can be cloned in lieu of the polyhedrin promoter and polyhistidine tag using any of the unique restriction endonuclease sites (SmaI, XbaI, EcoRI, NotI, PstI, BglII) located in the MCS. Proteins are expressed under control of strong very late polyhedrin promoter in the polyhedrin site of baculovirus DNA. Affinity purification of the expressed proteins can be achieved using Ni-NTA (nickel-nitrilotriacetic acid) resin (QUIAGEN). If desired, polyhistidine tag can be cleaved with thrombin at thrombin cleavage site (LVPRGS) encoded at nucleotides 4174-4191 of the vector. Unique SpeI site can be used to obtain 6xHis-tagged proteins without the thrombin site. Unique BamHI site can be use to obtain proteins without polyhistidine tag. Positions of phF and mR primers, which are useful for sequencing the insert/vector junctions, are shown on the DNA sequence as arrows.  
  
    
    
  
  If you have not worked with baculovirus expression system, also referred to as baculovirus expression vector system or BEVS, you can get a quick update at TECHNOLOGY. Go to BACULOVIRUS TUTORIAL for simple on-line instructions on baculovirus transfection, propagation of recombinant baculoviruses and recombinant baculovirus protein expression studies.

Click on PRODUCTS to view the entire list of recombinant baculovirus-related products.  



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Thanks
Anna

[pDNA]

I've checked the sequence and to my point of view since the Polyhedrine promoter is the only promoter upstream the MCS there should be no problem for E. coli (you can check the file attached!).

What is most curious to me is that you do not  get any colonies ...not even background.

I would recommend:
1) Do a ligation with only your digested insert or vector only ...just to check if these both ends are digested properly ...you should get a ladder with all the possible self-ligation products (dimers,trimers and so on ...looks like a DNA marker) ...for example if you only get the dimer only ...then you know that the digest with one of your enzymes was not successful

2) Check your fragment size and concentration prior to ligation ...to make sure you are using the correct stuff.
3) If you prepared your insert by PCR check your primers for the addition of 5' nonsense nucleotides in front of the restriction site ...for more details check this site.
4) A question: Do you have the feeling that your insert that you are trying to clone could be toxic to e. coli or does it only work in insect cells?
5) Concerning your cloning strategy: You stated that you use BglII and BglII and Xba since you have two inserts? You are cloning them at once or sequential? I do not really get it ...maybe you can describe more clearly what you have in mind?  

Good luck!

Regards,
p

Attached Thumbnails

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Edited by pDNA, 19 June 2010 - 03:31 AM.

[ranvi]

Hi P,

we were trying to swap the toxic gene to pAB vector. Inorder to have the compatible ends with pAB we had to do the mutation in our pXLG vector which had the toxic gene, becos of repetitive sequencein our pXLG vector the mutation was very difficult and we made it to work,  Mutation was to introduce BglII and XbaI and to remove bglII. During the mutation  there were some deletion,  and also introduced addnl bglII site in one of the insert.  so instead of cutting with BglII and Xbal we had to just linearize one of the vector(where toxic part to be sliced from) with BglII and that gave 2 fragments, which we were trying to subclone into pAB.

The other cterminal insert when mutated it gave Xbal and BglII and we had to clone that into pABdigested with XbaI and BglII

I too think it is the digestion is not complete,
when i tried to BglII and xbaI (NEB) double digeted sometimes i get undigested part. So I went for sequential digestion. When I put excess it gives star activity. Finally I managed to get to a complete digestion  

After gelpurification I run everything on the gel before ligation it looks very clean and digested.

I did 2hrs ligation, overnight ligation. 20hrs ligation.

Control : Vector after SAP treatment did not give colonies (BglII cut pAB)
             VEctor  Bglii and XbaI cut did not give colonies so thats good.

I also attached my digested vector and insert went for ligation
is Any thing  look not clean from the gel pic?

Thank you for the continous help I hope I will be able to make it to work

Attached Thumbnails

• 

[ranvi]

In the gel from the right after Marker (WHITE Pict)
1. the vector linearized with BglII(pAB NT) NOt gel purified just pcr cleaned

2. Insert for NT
3. Gel purified Ct (BglII XbaI cut pAB)
4. pcr cleaned not gelpurified ( BglII XbaI cut
5. CT Insert

[pDNA]

your gel seems to be fine!

i must confess i do not really get what you are trying to do ...but sometimes it is hard to explain not being in a face-to-face situation.

i would recommend setting up a ligation reaction with only the two inserts and without vector ...and load those two reactions on an agarose gel to check if both inserts are digested properly (you can post that gel pic here if you want to).

If you vector backbone is really dephosphorylated 100% (since you do not get colonies) it is essential that your insert is correctly digested ...so you have to check this first.

Further it would be interested who you introduced the formermentioned mutations in your vetor backbone? By PCR mutagenesis? ...maybe you destroyed some essential part in the origin of replication of your vector and therefore you are unable to generate colonies?

Good luck!

Regards,
p

[ranvi]

In the Gel The 2 bands in the lower marker range around 3.8 to4kb (it ispromega 1kb ladder )they are the two inserts one is from Nterminal another is cterminal almost the same size

All I am trying to  do is swap the toxic gene with  other essentail portion of the vector which is created as destination vector for GAte way cloning for mammalian expression into the baculovirus vector so that we  can  transfect the naked dna into baculovirus system once they spit the naked dna with viral component that can be injected into mammalian cells with out introduction of any transfection reagents like PEI or other liposome regents which we are experiencing much difficulties (Have you heard of BAC MAM system thats what we are trying to do)
When we cut the insert we left the ORI part so it doesnt have any.
Thanks
Anna

[ranvi]

For cloning my insert into BAC system we did nothave sites compatible to the Baculovirus vector. In the MCS of pAB Vector we chose XBaI and BglII to clone our insert. Since our insert doesnt have this we had to introduce by mutation ( I did point mutation  although it should be st fwd procedure it was difficult due its repetitve seq pattern of our pXLG vector)

[pDNA]

why you did not amplify your insert using PCR and add the restriction sites on the PCR primers? ...this would be more straightforward?

Regards,
p