Clonig Problem - (Sep/02/2007 )
My problem is: I have digested my vector with BamHI and Hind III to liberate a fragment of 800pb, and now I have to re-ligate the vector, ´cause I need to open it with another pair of enzymes. When I religate the vector and checked the colonies they still have an 800pb fragment! What should I do??? Please help me!!!!!
if you need to reopen it by 2 others enzymes where are they located regarding bam h1 and hind III ? i can't get why you don't digest directly with these
if the vector has been digested by BamHI and HindIII, the resulting sticky ends are not complementary. Thus the resultings ends are will not ligate.
Could you please give us more information to work on. Can you tell us in detail what exactly you want to do. And perhaps provide a restriction map of your plasmid.
For as Fred_33 has pointed out, can you not use the two other restriction enzymes mentioned and ligate your desired insert directly without making an intermediate empty vector.
ok! here i go!
in details: My vector is pt7-177, it´s an rnai vector for t.brucei, it has a frgment of gfp (800 pb) between hindIII and bamHI sites. So I have to take out the gfp fragment to clone my fragment.
the vector is like that : T7 promoter- bamHI- gpf- hindIII- xhoI- T7 promoter. My fragment came from another vector and it was liberated out of this vector with xho and hind. I would like to use this fragment to clone in pt7-177. The fragment has to be between the 2 T7 promoters to induce double strand RNA. So I have to take out the gfp fragment (with bamhI and hind III) religate the vector, and redigest with xho and hind to clone my insert. When i digested it with bamHI and hindIII i saw the gfp fragment and cut the vector band, purified, and religated it. But all the colonies still have the gfp fragment when I digest them with bamHI and Hind III. Bam HI and Hind are not compatible, the ends will never religate? Can I use some enzyme like Klenow?
Do you think, there is a way that I could use my fragment with xho-hind? Or should I do another primer to clone in this vector? like a primer with BamHI...
BamHI and HindIII is not compatible, "if" the overhangs do religate, then you will not be able to reuse them, as in, cannot cut with BamHI and/or HindIII anymore. That defeats the purpose, I think. Maybe reclone your insert (PCR it out) with BamHI/HindIII or BamHI/XhoI. I have never work with this vector and not familiar with what you want to produce, but take from your exp. design, I guess that the direction of the ligated fragment is not important? Can we use blunt end in this case?
I can see three ways you may go about this.
As you have already mentioned, you can use a pair of primers and via PCR add a BamHI and HindIII site to your insert fragment. This would be the fastest and easiest
2) Partial fill in
XhoI and BamHI when partialled filled in with 2 appropriate basepairs and klenow, form compatible ends. So you will have to first digest your vector with BamHI. Conducted a partial fill in. Clean up the DNA, Then digest the vector again with HindIII. Finally gel purify. The same thing happens with the insert. First digest with XhoI. Then partial fill in. Then a clean up step. Then digest with HIndIII. And finally gel purify.
This method if abit of work. But you can still use the XhoI/HIndIII restriction sites on the insert after a fashion.
3) Blunt ending
As Almasy has suggest you may blunt end both your insert and vector by fully filling in the restriction site overhangs by treating the DNA with dNTPs and klenow.
This method is fast. But the ligation can be difficult.
Of the three methods available, I would do as you have already suggested and make a primer pair to clone in the insert. As remember to buy one more primer, so you can do colony PCR.
Thanks so much! What do tou think about this:
I have this vector (pt7177) opened with BamHI and HindIII, the band is purified, do you think I could digest the opened vector with XhoI treat with phosphatase,purify the band and then ligate my insert (which has Xho and Hind)? Then I would purify the band again, treat with klenow and religate...
Much easier than any of this is to design two primers with the RE cut sites you want which prime the amplification of the entire vector backbone. Keep what you want, eliminate what you don't. One PCR reaction, cut, ligate, done.
I have to agree with Phage434. Keep it simple.
As a rule with molecular biology, the more steps you need to conduct the more likely it is going to fail. That is why so many people have problems with ligation work. The process requires several steps. Get one step wrong or several step less then perfect and the ligation just doesn't work.
Ok you all have convinced me! I ordered the primer with BamHI!
For a while ( until I get the primer in hands) I´ll try the blunt end! If I clone anything I´ll tell you!
Thanks a lot for all the answers, and for replying so fast!