5 replies to this topic

[Juliasarmoire]

Hi, I'm really new at everything... and the more I read the more confused I seem to get I'm currently doing my master's thesis (in molecular biology) and my current project is to purify a protein (later to get an antiserum)... till this point everything has worked out fairly well, but now I have one question for someone who hopefully knows something.

I have used gel purifying after PCR (with QIAEX Gel Extraction kit), but the yield was so poor that I changed to use Nucleospin Extract II kit to purify the PCR products, the yield went up. I'm using pET28a plasmid with two restriction enzymes (Sal I and EcoR I), again after digestion I have purified the plasmid with QIAEX Gel Extraction kit, but the yield is so bad that I have do something else... I only get one clear band, so it seems that most of the plasmid is cut in the right way, so I was wondering can I use Nucleospin Extract II kit (for PCR) to purify the plasmid too? I know that in that way I wouldn't get rid of the undigested plasmids, but I'm willing to do anything to get the yield up... So is the kit strictly for PCR or can you use it for plasmid too?

Also is it necessary to get give the plasmid a CIP treatment even I have used two different restriction enzymes? It seems that people have different opinions... thank you for any help

[Warren]

Juliasarmoire, on Sep 10 2009, 06:36 AM, said:

Hi, I'm really new at everything... and the more I read the more confused I seem to get I'm currently doing my master's thesis (in molecular biology) and my current project is to purify a protein (later to get an antiserum)... till this point everything has worked out fairly well, but now I have one question for someone who hopefully knows something.

I have used gel purifying after PCR (with QIAEX Gel Extraction kit), but the yield was so poor that I changed to use Nucleospin Extract II kit to purify the PCR products, the yield went up. I'm using pET28a plasmid with two restriction enzymes (Sal I and EcoR I), again after digestion I have purified the plasmid with QIAEX Gel Extraction kit, but the yield is so bad that I have do something else... I only get one clear band, so it seems that most of the plasmid is cut in the right way, so I was wondering can I use Nucleospin Extract II kit (for PCR) to purify the plasmid too? I know that in that way I wouldn't get rid of the undigested plasmids, but I'm willing to do anything to get the yield up... So is the kit strictly for PCR or can you use it for plasmid too?

Also is it necessary to get give the plasmid a CIP treatment even I have used two different restriction enzymes? It seems that people have different opinions... thank you for any help

The short answer is, I think you could probably use your kit to "purify" your plasmid.  I don't have experience with that kit, but if it can purify PCR products as big as your plasmid, it'll probably work for the plasmid as well.  Another short answer as to phosphatase treatment -- if I can avoid it I do, but basically it depends on how well your enzymes cut -- if there is a percentage of plasmid cut by only one of the enzymes than the self-ligation will outcompete the insert and you will have alot of vector only bacteria colonies.  If you can do blue/white screening this sometimes doesn't matter.  Just try it both ways, your experiment is simple enough.

But I am very curious how you are determining "yield" and why you are getting hung up on it?  The type of cloning you are doing is about as easy as you can do, two different cutting sites on the insert and vector, you won't need much to make this work.  The qiagen gel-purification should give you tons -- I have used this system for 14+ years and rarely have any problems.  Of course the gel system is only going to yield at most what you load on the gel, and you always lose a little.  Are you rerunning some on a gel to determine yield?  The reason I ask is I wonder how your spin columns seem to work so well but your gel extractions don't.  Your spin columns may be allowing primers, dntps, primer dimers, etc to end up in your final product giving you an apparent higher "yield" if you are trying to measure absorbance or something.  I would at least double check on a gel.  With what you have currently, I would: spin-purify your PCR product then digest, digest your vector, run them both on a gel and gel-purify, then do ligation (with or without phophatase-treated vector).  You should get your desired clone no problem.  You could waste alot of your graduate study time trying to get the "best yield" you can.  Good luck!  Warren..

[Juliasarmoire]

Warren, on Sep 11 2009, 07:44 AM, said:

Juliasarmoire, on Sep 10 2009, 06:36 AM, said:

Hi, I'm really new at everything... and the more I read the more confused I seem to get I'm currently doing my master's thesis (in molecular biology) and my current project is to purify a protein (later to get an antiserum)... till this point everything has worked out fairly well, but now I have one question for someone who hopefully knows something.

I have used gel purifying after PCR (with QIAEX Gel Extraction kit), but the yield was so poor that I changed to use Nucleospin Extract II kit to purify the PCR products, the yield went up. I'm using pET28a plasmid with two restriction enzymes (Sal I and EcoR I), again after digestion I have purified the plasmid with QIAEX Gel Extraction kit, but the yield is so bad that I have do something else... I only get one clear band, so it seems that most of the plasmid is cut in the right way, so I was wondering can I use Nucleospin Extract II kit (for PCR) to purify the plasmid too? I know that in that way I wouldn't get rid of the undigested plasmids, but I'm willing to do anything to get the yield up... So is the kit strictly for PCR or can you use it for plasmid too?

Also is it necessary to get give the plasmid a CIP treatment even I have used two different restriction enzymes? It seems that people have different opinions... thank you for any help

The short answer is, I think you could probably use your kit to "purify" your plasmid.  I don't have experience with that kit, but if it can purify PCR products as big as your plasmid, it'll probably work for the plasmid as well.  Another short answer as to phosphatase treatment -- if I can avoid it I do, but basically it depends on how well your enzymes cut -- if there is a percentage of plasmid cut by only one of the enzymes than the self-ligation will outcompete the insert and you will have alot of vector only bacteria colonies.  If you can do blue/white screening this sometimes doesn't matter.  Just try it both ways, your experiment is simple enough.

But I am very curious how you are determining "yield" and why you are getting hung up on it?  The type of cloning you are doing is about as easy as you can do, two different cutting sites on the insert and vector, you won't need much to make this work.  The qiagen gel-purification should give you tons -- I have used this system for 14+ years and rarely have any problems.  Of course the gel system is only going to yield at most what you load on the gel, and you always lose a little.  Are you rerunning some on a gel to determine yield?  The reason I ask is I wonder how your spin columns seem to work so well but your gel extractions don't.  Your spin columns may be allowing primers, dntps, primer dimers, etc to end up in your final product giving you an apparent higher "yield" if you are trying to measure absorbance or something.  I would at least double check on a gel.  With what you have currently, I would: spin-purify your PCR product then digest, digest your vector, run them both on a gel and gel-purify, then do ligation (with or without phophatase-treated vector).  You should get your desired clone no problem.  You could waste alot of your graduate study time trying to get the "best yield" you can.  Good luck!  Warren..

Thank you I have continued using the QIAEX Gel Extraction kit for the plasmid, just in case (as I have been using just kanamycin plates and the kanamycin selection is in the plasmid, not in the insert, so undigested plasmids grow  in the plates too), but I might as well try to use the other kit too... "yield" has been measured by using the Nanodrop (so yes, absorbance). I have been loading over 1000 ng to the gel, but after cutting out the band and using the gel extraction kit I get something like 20-40 ng in total, so it feels the entire plasmid is gone and I have repeated it many times in the past week even having someone else checking that I'm doing each step right, but the plasmid is somehow gone. It's enough for the ligation as I noticed today, but it's still not much. I have to repeat that one step over and over again... so I'm not trying to get massive yield, but I have been trying to find way to get enough plasmid for more than one ligation reaction at a time Even at least I remember the entire QIAEX protocol by heart now

I have used spin-purify PCR producs then digest, digest the vector, spin-purify the PCR products again, run vector, gel-purify, ligation, transformation, plasmid purifying, then digestion again to run the products on the gel... this is an old project someone has tried and the ultimate problem is that the insert hasn't been in the right reading frame... out of the six samples I ran today, all has vectors and inserts, but all inserts are a bit different size... but I'm keeping my thumbs up that even one of them would have the right sequence I'm just trying to triple check that each step is right so that in ideal case everything would go fine at once... otherwise I probably will need to design new primers or something But thanks... it's interesting to be back at molecular biology after doing nothing else than neuroscience and measuring currents for the past year... it's coming back, slowly, but surely

[Warren]

[/quote]

Thank you I have continued using the QIAEX Gel Extraction kit for the plasmid, just in case (as I have been using just kanamycin plates and the kanamycin selection is in the plasmid, not in the insert, so undigested plasmids grow  in the plates too), but I might as well try to use the other kit too... "yield" has been measured by using the Nanodrop (so yes, absorbance). I have been loading over 1000 ng to the gel, but after cutting out the band and using the gel extraction kit I get something like 20-40 ng in total, so it feels the entire plasmid is gone and I have repeated it many times in the past week even having someone else checking that I'm doing each step right, but the plasmid is somehow gone. It's enough for the ligation as I noticed today, but it's still not much. I have to repeat that one step over and over again... so I'm not trying to get massive yield, but I have been trying to find way to get enough plasmid for more than one ligation reaction at a time Even at least I remember the entire QIAEX protocol by heart now

I have used spin-purify PCR producs then digest, digest the vector, spin-purify the PCR products again, run vector, gel-purify, ligation, transformation, plasmid purifying, then digestion again to run the products on the gel... this is an old project someone has tried and the ultimate problem is that the insert hasn't been in the right reading frame... out of the six samples I ran today, all has vectors and inserts, but all inserts are a bit different size... but I'm keeping my thumbs up that even one of them would have the right sequence I'm just trying to triple check that each step is right so that in ideal case everything would go fine at once... otherwise I probably will need to design new primers or something But thanks... it's interesting to be back at molecular biology after doing nothing else than neuroscience and measuring currents for the past year... it's coming back, slowly, but surely
[/quote]

If you are losing more than 95% of your DNA doing the gel extraction, something is wrong....I am suspicious of using this "nanodrop" system and whether you are getting a true reading -- in order for absorbance to work accurately you need to zero with the correct buffer for each sample and it should be relatively pure (thats why I don't like to use it to measure each step in manipulations like this but would rather estimate on a gel), for me absorbance is best used on pure preps, ie, usually your starting material.  Have you checked to see if it looks like you are really losing 95% of your sample on a gel?  

But the other thing that puzzles me is the "correct reading frame".....your PCR product should define this, ie you have specific primers with specific cleavage sites.  You shouldn't get multiple insert sizes unless you had multiple PCR products, in which case PCR optimization is the way to go along with, perhaps, gel purification of the correct size band.  Also based on experience, it is sometimes best to get new PCR primers (because they are so cheap) rather than force the ones you have to work (been there, done that!)  Good luck, warren..

[Juliasarmoire]

To me it looks and feels like I'm getting the plasmid out of the gel really nicely (I ran two bands, the one which I check out under the UV and the other one which I cut out, which is not exposed to UV and everything should be fine, in theory). It seems to be working, so we will see how it will work out... I will keep my thumbs up.

I also know about the correct reading frame... I don't know why it hasn't worked out in the past and why I seem to be having different inserts. While I ran the PCR products on the gel, I have one band, so I don't know what happens when I do digestion, ligation and transoformation to change it. I'm using human cDNA library as a PCR template (which makes things interesting as half of my current primers get totally wrong bands) and the protein which I'm working on is large (> 500 kDa). I'm only focusing into c-terminal and my current fragment should be about 500bp long... I have two different sets of primers close to each other, so I'm hoping that something will work out as the next step is to design new primers anyway as I need to get another 500bp fragment... to have more changes to get the antigen against the protein's c-terminus (which is the final step of my thesis). So in theory the primers seem to working fine... next week I will get the sequence results back and hopefully I will be smarter by then and hopefully the sequence is from the right protein and not something else... still in theory it should work, but it doesn't. I will also try to come up with new primers in the next week... hopefully with better and more specific results. Thank you again! And the good news is that even if this project won't work out, I can always write my thesis... with null results... even it would be nice to achieve something

[Warren]

Juliasarmoire, on Sep 12 2009, 02:12 PM, said:

To me it looks and feels like I'm getting the plasmid out of the gel really nicely (I ran two bands, the one which I check out under the UV and the other one which I cut out, which is not exposed to UV and everything should be fine, in theory). It seems to be working, so we will see how it will work out... I will keep my thumbs up.

I also know about the correct reading frame... I don't know why it hasn't worked out in the past and why I seem to be having different inserts. While I ran the PCR products on the gel, I have one band, so I don't know what happens when I do digestion, ligation and transoformation to change it. I'm using human cDNA library as a PCR template (which makes things interesting as half of my current primers get totally wrong bands) and the protein which I'm working on is large (> 500 kDa). I'm only focusing into c-terminal and my current fragment should be about 500bp long... I have two different sets of primers close to each other, so I'm hoping that something will work out as the next step is to design new primers anyway as I need to get another 500bp fragment... to have more changes to get the antigen against the protein's c-terminus (which is the final step of my thesis). So in theory the primers seem to working fine... next week I will get the sequence results back and hopefully I will be smarter by then and hopefully the sequence is from the right protein and not something else... still in theory it should work, but it doesn't. I will also try to come up with new primers in the next week... hopefully with better and more specific results. Thank you again! And the good news is that even if this project won't work out, I can always write my thesis... with null results... even it would be nice to achieve something

A couple of things:  >500kda?!?!  Wow, what protein are you working with???  I thought dystrophin was the biggest human protein, I am very interested in what protein is so large!

Get new primers:  its the easiest way to solve everything, and strongly consider different restriction sites in the vector.

The problem in your sequence of events is doing a restriction digest on your PCR product, then doing a spin purification.  You should gel purify after digestion.  If you are getting all different size inserts (and assuming a single band after PCR), your probably getting alot of star activity from one or both enzymes, resulting in multiple "products" -- if you run a gel you should see those as a smear or possibly discrete bands.  EcoRI has a lot of star activity if you "over digest" or don't have perfect conditions.  I think gel purifying your insert after digestion is extremely important in this case.  

Warren..