6 replies to this topic

[vetticus3]

Hey all,
I am so seriously screwed.  
I'm still trying to clone my 300bp product into pBSKS+.   Previously, I had used unphosphorylated vector , and had a lot of background, but with blue-white selection,  I had a good shot of picking the colonies with the insert.  Anyway....use CIAP, and the insert wasn't ligating.  Went back to using unphosphorylated vector, 3:1 molar ration...yadayadayada....same as before....Now what has previously worked, doesn't.  Getting really weird results as well, with the cut miniprep in some colonies looking as though they're not cut, and staying at ~10 000bp mark.  pBSKS+ is only 2.9Kb.  And my insert, 300bp.  
I figured, maybe the enzymes aren't recognising the site... I was using BamHI and EcoRI,  went outside these two with XbaI and HindIII.  And there was insert in some of the clones.  I expect bands at ~300bp....BUT i'm getting bands at ~800bp, and 2000bp.  Where the hell are they coming from?  
I haven't used another vector, because i can't get my paws on one, yet...

So, went back and recut some vector with BamHI and EcoRI (i'm not purifying this.... could this be the problem?)  Went back and PCRed my insert with Pfu, purified it, cut with BamHI and EcoRI....Ligate, 3:1 molar ratio, room temp overnight.... Ethanol precipitate....electro transform....plate.... blue white selection.... pick positive colonies, miniprep.....double digest again, with BamHI and Eco RI....and also take another sample and double digest with XbaI and HindIII....run the gel.... yeah, some work...but really inefficient, and an ~800bp product, ~20000, product, as well as my ~300 insert, and the ~3000 bp vector in the sample I cut with XbaI and HindIII.

Can someone please tell me where i'm going wrong?  
Other people in my lab don't purify their vector, and they had no problems.
I don't get it.  I need help.... please.
Oh, does TA cloning work well with Pfu instead of Taq?

Vetticus

Edited by vetticus3, 11 October 2004 - 04:49 PM.

[caro]

Hi Vetticus

TA cloning only works when there is an overhang, if you use pfu, you can make an ekstra mix with taq polymerase and add it to the tubes after the pcr, and give it 10' elongation, then the product has the overhang for TOPO TA cloning.
Are you sure your product is 300 bp? You can purify it from gel with Amershams Gel band DNA purification kit.
Good Luck
Caro

[chandima]

dear freind
I am having the same problem i don't know for how long,  i get loads of blue but no white colonies, Now I am going to try TOPO cloning. That seems a good system
good luck
chandima

[musicflower]

chandima, on Oct 12 2004, 12:43 AM, said:

dear freind
I am having the same problem i don't know for how long,  i get loads of blue but no white colonies, Now I am going to try TOPO cloning. That seems a good system
good luck
chandima

what is TOPO cloning?can you explain it to me?i dont know.thank you.

[jadefalcon]

@ musicflower

TOPO (TA) cloning basically means that there's a single base T overhang at the commercially available LINEAR vector AND there's a topoisomerase covalently bound to the respective ends. when your doing your pcr with taq pol, it will generate a single base A overhang at the ends of the PCR product that's compatible with the T at the vector. so you have some kind of sticky end cloning and a topoisomertase that'll keep the DNA in place for the ligase to stitch it together.

Therfore TOPO TA cloning. if you want to read the brochure from one manufacturer:

http://www.invitroge...Cloning_bro.pdf

mike

[tfitzwater]

1. Check out my ligation and transformation protocol at http://micro.nwfsc.noaa.gov/protocols/.  It is written for 100 bp inserts, but scales easily for larger ones.  

2. Pfu leaves blunt ends.  No A-overhang for the TA cloning kit to work with.  

3. Hu has shown that the extended nucleotide is not always an A residue, and is dependent on the specific nucleotide present on the 5’ ends of the PCR DNA. (Hu, 1993 DNA and Cell Biology 12 (8) 763-770.) Also see Hite, J., Eckert, K. A., Cheng K. C., 1996.  Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)nd(G-T)n microsatelite repeats.  Nucleic Acid Res. 24:  2429-2434.

If the 5’ nucleotide of the primer is an A, then Taq modifies the 3’ end to - T, + A.
If the 5’ nucleotide of the primer is a C, then Taq modifies the 3’ end to +G >  + A > +C.
If the 5’ nucleotide of the primer is a G, then Taq modifies the 3’ end to + A > +C.
If the 5’ nucleotide of the primer is a T, then Taq modifies the 3’ end to  + A.

4. Inserts of less than 1000 bp do not always generate white colonies.  

5. Check your digestions with controls of single cuts.  You can remove the MCS fragment from the vector with a Microcon cartridge rather than gel purification.  Be sure the DNA is as clean as possible before ligation.

[vetticus3]

you people rock.