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is my cloning strategy right - (Nov/27/2006 )

Hello all, Please comment on my cloning strategy......
I got one gene in pBIN PLUS vector cloned on Sal1 position. GFP gene is cloned inside this gene on NcoI and BamHI site. I want to replace this GFP with mRFP.
I am planning to digest out these 2 genes from pBINPLUS from SalI position and reclone to pUC19 since pBINPLUS got 3 Nco1 and 2 BamHI site. After cloning to pUC19 I will digest out GFP fragment from NcoI-BamHI site and replace with PCR amplified mRFP. This casste will again digest out from SalI site and reclone to pBINPLUS SalI site. This is my strategy.
experts, kindly suggest is there any better strategy.
I am worried about the direction of ligation on SalI position.
Please help, I am under pressure, Thanks in advance

-sse-

Logically your ligation strategy is sound....
However I am very warry of using SalI in my cloning strategy. This restriction enzyme has a well dserved reputation in my lab of causing trouble. So be advice, there maybe shoals in your future.

Aside from that, don't be too worried about the non-directionality of the mRFP, do a restriction digestion on your clones to look for a clone that is in the correct orrientation... 50/50... so half of the clones that take up mRFP will be right.

Without a more detailed map, I can not propose a better and safer strategy. However, if your plasmid is about 5kb, you may want to consider this....



This strategy amplifies your vector, using primers that miss out the GFP gene. It would help if you could cut within the GFP gene, linearising the vector and making the PCR more efficient. (avoids tertiery structure problems... which can be a killer)

The main benefit of the above stratergy is that it is potentially faster and avoids use of SalI digest... which I believe will lead to tears. Minor benefit is that you don't have to worry about orrientation problems.

Major draw back... vector PCR... not for the faint hearted, vector size limit. However if your vector is not too large, then it is not a problem. Needs a good proof reading enzyme to avoid errors...counter balance by the fact that errors in the vector backbone has no effect.

For stuff around 5kb, I recommend KOD hifi, more then 7kB... phusion, takara's long template (can't remember the name), KOD long template.. etc ....all do fine...

-perneseblue-

QUOTE (perneseblue @ Nov 28 2006, 01:29 AM)
Logically your ligation strategy is sound....
However I am very warry of using SalI in my cloning strategy. This restriction enzyme has a well dserved reputation in my lab of causing trouble. So be advice, there maybe shoals in your future.

Aside from that, don't be too worried about the non-directionality of the mRFP, do a restriction digestion on your clones to look for a clone that is in the correct orrientation... 50/50... so half of the clones that take up mRFP will be right.

Without a more detailed map, I can not propose a better and safer strategy. However, if your plasmid is about 5kb, you may want to consider this....



This strategy amplifies your vector, using primers that miss out the GFP gene. It would help if you could cut within the GFP gene, linearising the vector and making the PCR more efficient. (avoids tertiery structure problems... which can be a killer)

The main benefit of the above stratergy is that it is potentially faster and avoids use of SalI digest... which I believe will lead to tears. Minor benefit is that you don't have to worry about orrientation problems.

Major draw back... vector PCR... not for the faint hearted, vector size limit. However if your vector is not too large, then it is not a problem. Needs a good proof reading enzyme to avoid errors...counter balance by the fact that errors in the vector backbone has no effect.

For stuff around 5kb, I recommend KOD hifi, more then 7kB... phusion, takara's long template (can't remember the name), KOD long template.. etc ....all do fine...

Hello perneseblue,
Thank you very much for ur brilliant idea. U really spend lot of time with nice figure and all
Hope u wont mind if I ask few more doubts since I am not so femiliar in this field.
Actually pBINPLUS + gene+gfp is around 15.5 kb in size. Can I proceed as u suggested?
We got pfu from Fermentas in our lab. Can I use it?
Even if I generate 2 new restriction sites on mRFP and gene ends would is interfere with reading frame and all, if the sites add up or reduce bases? I mean the reading frame of expressing transgene?

please please help

-sse-

QUOTE (sse @ Nov 28 2006, 08:53 PM)
Hello perneseblue,
Thank you very much for ur brilliant idea. U really spend lot of time with nice figure and all
Hope u wont mind if I ask few more doubts since I am not so femiliar in this field.
Actually pBINPLUS + gene+gfp is around 15.5 kb in size. Can I proceed as u suggested?
We got pfu from Fermentas in our lab. Can I use it?
Even if I generate 2 new restriction sites on mRFP and gene ends would is interfere with reading frame and all, if the sites add up or reduce bases? I mean the reading frame of expressing transgene?

please please help


A picture is worth a thousand words. It is a much better way of getting the point across. wink.gif

pBINPLUS + gene+gfp = 15.5kb

My mistake, I did not realise pBINPLUS was such a large plasmid. Unless you have experience with long range PCR, it would be best not to procced with my approach. 15.5kb is quite long, and may need optimisation of the long range PCR protocol, which in the worse case senario can take a lot of time.

I have not used pfu from fermentas before, so I can not comment.

The restriction sites, if design wrongly can affect the reading frame. So introducing such sites must be done with care. Always test your sequence on a programe like VectorNTI or similar to check that the reading frame has not been changed. (But practically nearly all restriction enzymes are 6bp cutter... just have to be careful not to introduce stop codons.)

So back to the drawing board....

I have one question.... is your gene cut in half by GFP, or is the GFP merely added to it's C terminal end? Is there any sequence of importance behind the GFP that is not part of the blank pBINPLUS?

If the GFP is merely a fusion to your gene's C terminal. And you have access to empty pBINPLUS you might try this;



NCBI pBINPLUS

This is a three way ligation. According to NCBI, the blank pBINPLUS has LacZ, thus insertion into the Multiple Cloning Site can be screened by colour testing. The restriction enzyme A and B must not cut your GENE. B and C do not cut mRFP while restriction site A and C are restriction sites in the MCS of pBINPLUS. You'll have to check if such a combination is possible.

So provided you have access to empty pBINPLUS and the restriction site are favourable, going by PCR and 3 way ligation... is ~3 times faster then restriction digest method.

The benefits of this method : Speed. And avoids SalI digest
Drawbacks: requires access to blank pBINPLUS, needs resequencing of GENE. (mRFP will naturally need sequencing.)

If my calculation are correct your gene is only 2.3kb, which is nice, small and easily cloned by any good proofreading polymerase.

PS. Before I forget, I am assuming you are using supercompetent cells (like Genehog ...). Ligation with plasmids this size is quite poor, so company made cells are required.

-perneseblue-

QUOTE (perneseblue @ Nov 29 2006, 01:37 AM)
QUOTE (sse @ Nov 28 2006, 08:53 PM)

Hello perneseblue,
Thank you very much for ur brilliant idea. U really spend lot of time with nice figure and all
Hope u wont mind if I ask few more doubts since I am not so femiliar in this field.
Actually pBINPLUS + gene+gfp is around 15.5 kb in size. Can I proceed as u suggested?
We got pfu from Fermentas in our lab. Can I use it?
Even if I generate 2 new restriction sites on mRFP and gene ends would is interfere with reading frame and all, if the sites add up or reduce bases? I mean the reading frame of expressing transgene?

please please help


A picture is worth a thousand words. It is a much better way of getting the point across. wink.gif

pBINPLUS + gene+gfp = 15.5kb

My mistake, I did not realise pBINPLUS was such a large plasmid. Unless you have experience with long range PCR, it would be best not to procced with my approach. 15.5kb is quite long, and may need optimisation of the long range PCR protocol, which in the worse case senario can take a lot of time.

I have not used pfu from fermentas before, so I can not comment.

The restriction sites, if design wrongly can affect the reading frame. So introducing such sites must be done with care. Always test your sequence on a programe like VectorNTI or similar to check that the reading frame has not been changed. (But practically nearly all restriction enzymes are 6bp cutter... just have to be careful not to introduce stop codons.)

So back to the drawing board....

I have one question.... is your gene cut in half by GFP, or is the GFP merely added to it's C terminal end? Is there any sequence of importance behind the GFP that is not part of the blank pBINPLUS?

If the GFP is merely a fusion to your gene's C terminal. And you have access to empty pBINPLUS you might try this;



NCBI pBINPLUS

This is a three way ligation. According to NCBI, the blank pBINPLUS has LacZ, thus insertion into the Multiple Cloning Site can be screened by colour testing. The restriction enzyme A and B must not cut your GENE. B and C do not cut mRFP while restriction site A and C are restriction sites in the MCS of pBINPLUS. You'll have to check if such a combination is possible.

So provided you have access to empty pBINPLUS and the restriction site are favourable, going by PCR and 3 way ligation... is ~3 times faster then restriction digest method.

The benefits of this method : Speed. And avoids SalI digest
Drawbacks: requires access to blank pBINPLUS, needs resequencing of GENE. (mRFP will naturally need sequencing.)

If my calculation are correct your gene is only 2.3kb, which is nice, small and easily cloned by any good proofreading polymerase.

PS. Before I forget, I am assuming you are using supercompetent cells (like Genehog ...). Ligation with plasmids this size is quite poor, so company made cells are required.

Hi perneseblue,
Thanks again for your consideration.
Actually GFP is in almost middle part of the transgene. Today whole the day I was trying to apply ur strategy to my cloning but unfortunately I found that its very didficult to find a 2 unique enzyme sites because pBINPLUS, transgene, GFP and mRFP all together covers almost all the main iction sites. Also I found that it will force us to spend quite a good amount of money too and its going to be waste because our lab is not more to Molecular Biology. But I am sure that I will make use of your strategy in future.
As u said I am already facing problem with SalI ligation. There is no any problem with digestion. pUC19 is getting digested and I am digesting out the GFP transgene fragment too. But I am finding it really dificult to ligate transgeneGFP fragment to pUC19. I tried 1.1, 3.1, 1.3, 1.5, insert vector ratios but no use. Could it because of problem with dephosphorylation step? Could you please give some hints to improve my ligation efficiency? Thank you very much

-sse-

QUOTE (sse @ Nov 29 2006, 06:18 PM)
Hi perneseblue,
Thanks again for your consideration.
Actually GFP is in almost middle part of the transgene. Today whole the day I was trying to apply ur strategy to my cloning but unfortunately I found that its very didficult to find a 2 unique enzyme sites because pBINPLUS, transgene, GFP and mRFP all together covers almost all the main iction sites. Also I found that it will force us to spend quite a good amount of money too and its going to be waste because our lab is not more to Molecular Biology. But I am sure that I will make use of your strategy in future.
As u said I am already facing problem with SalI ligation. There is no any problem with digestion. pUC19 is getting digested and I am digesting out the GFP transgene fragment too. But I am finding it really dificult to ligate transgeneGFP fragment to pUC19. I tried 1.1, 3.1, 1.3, 1.5, insert vector ratios but no use. Could it because of problem with dephosphorylation step? Could you please give some hints to improve my ligation efficiency? Thank you very much


The curse of SalI- cuts but DNA doesn't ligate..

Well, what are your dephosphorylation conditions? How much DNA (ng), size of DNA (bp), number of units of CIP added, final reaction volume and buffer used?

NEB's rule of the thumb is 0.1U CIP dephos 1pmol DNA, in 1 hr, at 37 Celsius within a reaction volume of 50ul

Over dephosphorlyation will degrade the overhangs.

Also make sure your T4 ligase and T4 ligase buffer is still working. Both components do not tolerate freeze thaw cycles well, with T4 ligase going bad the quickest. The ligase buffer should have a very strong smell of DTT. If the smell is gone, so has the buffer.

Also be aware of the solutions of vector and insert are suspended in. If they are suspended in TE or any EDTA containing buffer, percipitate the DNA and resuspend in sterile distilled water. EDTA is a strong inhibitor of the ligation reaction. EtOH is also another inhibitor, make sure none of these compounds get into your ligation reaction mix.

If you are not already using quick ligase buffer, you might consider getting some. The only difference between quick ligase buffer and regular ligase buffer, is the presence of 10% PEG6000 (final concentration). 10% PEG6000, make the ligation reaction more efficient. ... down side, is that PEG is a strong inhibitor of efficient bacteria transfection and thus must be removed. PEG is soluble in EtOH, so it was be washed off by EtOH percipitation followed by 70% EtOH wash.

Since this is SalI , I would suggest ligating longer then you would normally do.

Improved efficiency can also be helped abit by increasing your vector-insert concentration... ie more vector-insert in a smaller volume.

And lastly, you can do your ligation at different temperatures.... 16 Celcius or 4 Celsius. Ligating a lowered temperatures on some occassions help.

Well, best of luck. If there is no other way aside for using SalI, it is going to be a bumpy ride.

-perneseblue-

QUOTE (perneseblue @ Nov 29 2006, 09:27 PM)
QUOTE (sse @ Nov 29 2006, 06:18 PM)

Hi perneseblue,
Thanks again for your consideration.
Actually GFP is in almost middle part of the transgene. Today whole the day I was trying to apply ur strategy to my cloning but unfortunately I found that its very didficult to find a 2 unique enzyme sites because pBINPLUS, transgene, GFP and mRFP all together covers almost all the main iction sites. Also I found that it will force us to spend quite a good amount of money too and its going to be waste because our lab is not more to Molecular Biology. But I am sure that I will make use of your strategy in future.
As u said I am already facing problem with SalI ligation. There is no any problem with digestion. pUC19 is getting digested and I am digesting out the GFP transgene fragment too. But I am finding it really dificult to ligate transgeneGFP fragment to pUC19. I tried 1.1, 3.1, 1.3, 1.5, insert vector ratios but no use. Could it because of problem with dephosphorylation step? Could you please give some hints to improve my ligation efficiency? Thank you very much


The curse of SalI- cuts but DNA doesn't ligate..

Well, what are your dephosphorylation conditions? How much DNA (ng), size of DNA (bp), number of units of CIP added, final reaction volume and buffer used?

NEB's rule of the thumb is 0.1U CIP dephos 1pmol DNA, in 1 hr, at 37 Celsius within a reaction volume of 50ul

Over dephosphorlyation will degrade the overhangs.

Also make sure your T4 ligase and T4 ligase buffer is still working. Both components do not tolerate freeze thaw cycles well, with T4 ligase going bad the quickest. The ligase buffer should have a very strong smell of DTT. If the smell is gone, so has the buffer.

Also be aware of the solutions of vector and insert are suspended in. If they are suspended in TE or any EDTA containing buffer, percipitate the DNA and resuspend in sterile distilled water. EDTA is a strong inhibitor of the ligation reaction. EtOH is also another inhibitor, make sure none of these compounds get into your ligation reaction mix.

If you are not already using quick ligase buffer, you might consider getting some. The only difference between quick ligase buffer and regular ligase buffer, is the presence of 10% PEG6000 (final concentration). 10% PEG6000, make the ligation reaction more efficient. ... down side, is that PEG is a strong inhibitor of efficient bacteria transfection and thus must be removed. PEG is soluble in EtOH, so it was be washed off by EtOH percipitation followed by 70% EtOH wash.

Since this is SalI , I would suggest ligating longer then you would normally do.

Improved efficiency can also be helped abit by increasing your vector-insert concentration... ie more vector-insert in a smaller volume.

And lastly, you can do your ligation at different temperatures.... 16 Celcius or 4 Celsius. Ligating a lowered temperatures on some occassions help.

Well, best of luck. If there is no other way aside for using SalI, it is going to be a bumpy ride.

Hello perneseblue, great to hear from u again,
I think the main problem lying in my dephosphorylation step because I was not much concerned about dephosphorylation unit stuff. I am using Fermentas CIAP and I will definitely follow theit unit recommendation. Really there were so many useful tips for me and I am really more confident now. I will let u know the progress. Thank u very much. C U soon

-sse-