10 replies to this topic

[mahsa]

hey all
I have got a ligation reaction which is a bit of mystery and have got me into a real loop for sometime now, I believe what I have to do is to increase the ligation efficiency. the ligation itself dose not look to be extraordinary, I have to T/A clone a taq treated ap.7400 bp fragment into a vector with T overhangs, say pTZ57R or pGEM(promega). I cant figure out why it is not working.  
any hint or idea is deeply appreciated
Mahsa

[HomeBrew]

What do you mean by "taq treated"?  Did you treat a fragment with Taq to give it 3' A overhangs, or is the fragment a PCR product generated with Taq as the polymerase?  In either event, did you gel purify your insert DNA before using it in the ligation reaction?  Are you sure your recipient cells are competent?

[mahsa]

tnx a lot for quick response
sry for shortening the phrase, yes what I meant was taq polymerase treated. I have performed the PCR with a polymerase which possesses 3' to 5' proof reading activity, so I needed to add As to the fragment ends in order to get them to be ligated to the vector.
no matter what technique I use, what I get as a PCR result contains a non specific amplified 1500 bp fragment, so in order to have a specific insert fragment to ligate to the vector, although being exposed to EtB and UV reduces the ligation efficiency somewhat like 100 times, I have to gel purify the PCR amplified fragment then proceed with the ligation.
of competent cells, I am sure, because I have used the control plasmid to see if they work.

[HomeBrew]

Two suggestions -- you might want to try a blended polymerase preparation, like Invitrogen's Platinum PCR SuperMix High Fidelity (Cat. No. 12532-016).  This is a mix of Taq DNA polymerase and the proofreading Pyrococcus species GB-D polymerase, and provides ~6X higher fidelity than Taq alone (see here).  Additionally, the majority of fragments amplified with this mix have a 3-A' overhangs, so you wouldn't have to try and add them.  And, because it's a supermix, there's less pipetting involved -- the mix already contains dNTPs, Mg++, etc.

I don't work for Invitrogen, I've just used this product for years with good results.

To protect your DNA during gel purification, add 0.28 g/L (~1 mM) guanosine (e.g. Sigma G6752, FW 283.24) as a UV protectant to 1× TAE and stir with gentle heat until dissolved. Use this "sunblocked TAE" to cast the gel and as running buffer during electrophoresis -- see Grundemann, D. and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898-903. pdf here.

[mahsa]

HomeBrew, on 11 September 2010 - 07:07 AM, said:

Two suggestions -- you might want to try a blended polymerase preparation, like Invitrogen's Platinum PCR SuperMix High Fidelity (Cat. No. 12532-016).  This is a mix of Taq DNA polymerase and the proofreading Pyrococcus species GB-D polymerase, and provides ~6X higher fidelity than Taq alone (see here).  Additionally, the majority of fragments amplified with this mix have a 3-A' overhangs, so you wouldn't have to try and add them.  And, because it's a supermix, there's less pipetting involved -- the mix already contains dNTPs, Mg++, etc.

I don't work for Invitrogen, I've just used this product for years with good results.

To protect your DNA during gel purification, add 0.28 g/L (~1 mM) guanosine (e.g. Sigma G6752, FW 283.24) as a UV protectant to 1× TAE and stir with gentle heat until dissolved. Use this "sunblocked TAE" to cast the gel and as running buffer during electrophoresis -- see Grundemann, D. and E. Schomig. 1996. Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light. BioTechniques 21:898-903. pdf here.

thank you indeed for  very helpful tips, specially the research report.
what I use as a polymerase in PCR I perform is Expand High FidelityPLUS PCR System, dNTPack( Roche, Cat. No. 04 743 733 001) by which I actually get fragments that already have got the As to be T/A cloned, what I am trying to do is  increasing the chance of ligation between vector and insert by making sure that the majority of molecules have the As, like a double check point, the polymerase blend here also is X6 more accurate than taq.
BTW, I didn't think you work for Invitrogen   . the fact is Invitrogen has got very sophisticated products I have used some, they work absolutely grate.

[ElHo]

Is it possible that you might end up with AA-tails when additionally treating your PCR products with Taq? I´ve done TA-cloning using the expand high fidelity system whitout additional A-tailing and it worked. Maybe you could try to omit that step.

[mahsa]

ElHo, on 13 September 2010 - 12:44 AM, said:

Is it possible that you might end up with AA-tails when additionally treating your PCR products with Taq? I´ve done TA-cloning using the expand high fidelity system whitout additional A-tailing and it worked. Maybe you could try to omit that step.

dear EIHO
what I believe makes my whole T/A cloning a bit of a problem, is the insert size. It is a rather big one, 7377 bp. and the insert is gel-purified, so there is a chance that during the electrophoresis or other procedures, for ant reson, fragments have lost their terminal As. for this reason, I think Taq treatment would increase cloning chance.
Is AA overhang going to decrease cloning possibility? isnt it going to be filled in after transfecting into a host bacterial cell? i mena this is a nick, right? so it has to be filled by host. nah?

[ElHo]

I also don`t think gap filling would be a problem but I´m not sure about the ligation step (AA- and T-overhang). And you are right, about 7.4 kB is quite big, never cloned such a big fragment, sorry. Do you end up with a sufficient amount of purified insert after gel extraction?

[mahsa]

ElHo, on 15 September 2010 - 01:41 AM, said:

I also don`t think gap filling would be a problem but I´m not sure about the ligation step (AA- and T-overhang). And you are right, about 7.4 kB is quite big, never cloned such a big fragment, sorry. Do you end up with a sufficient amount of purified insert after gel extraction?

mmmm, I am gonna go with  no, but I have no complains. I use high pure pcr purification kit( roche) to purify fragments from gel, and the outcome is almost always disastrous, and I have to reduce the final volume by evaporation to get a reasonable concentration to proceed.
what do you do to purify the fragments from gel?

[ElHo]

Qiaquick gel extraction kit (Qiagen), but I´m also losing a lot with that one. But is the roche kit also suitable for gel extraction? I always thought it`s only used for PCR cleanup. Do you have to gel purify your PCR? Otherwise you could try to just purify your PCR product without gel extraction. Then you certainly will end up with more material to ligate.

[mahsa]

ElHo, on 15 September 2010 - 04:54 AM, said:

Qiaquick gel extraction kit (Qiagen), but I´m also losing a lot with that one. But is the roche kit also suitable for gel extraction? I always thought it`s only used for PCR cleanup. Do you have to gel purify your PCR? Otherwise you could try to just purify your PCR product without gel extraction. Then you certainly will end up with more material to ligate.

yea, the roche kit is designed for both pcr reaction purification and gel purification. I kinda have to extract the fragment form gel, 'cos there are unspecific pcr amplified fragments as well.