Please, some Tips for Maxiprep - (May/11/2006 )
Hi everyone.
I'm doing Maxi-Prep and I have achieved only one really successful. The rest of my attempts hasn't been really good, to be honest.
I wonder if anyone could answer the next questions and also give me some tips apart from those which are provided in the troubleshooting guide of Quiagen (I've tried some of them).
First of all, how much amount do you usually get when you do MaxiPrep (for 100ml of Lb, for ex)? (ug)
Secondly, I read somewhere else that some people always add Chloramphenicol regardless the plasmid they were amplifying.
How much OD of bacteria do you achieve before starting the column procedure? (I believe that I don't achieve the plateau phase)
Do you think there is any trouble if when defreezing your already transformed bacteria you put some of them directly in LB medium (with antibiotic) without using a plate.
Do you use Polypropile tubes to do MaxiPrep? Are there other type of tubes more convenient?
Please give me more suggestions.
Cheers
An apprentice
PS:
First of all, how much amount do you usually get when you do MaxiPrep (for 100ml of Lb, for ex)? (ug)
-->for low copy plasmids, up to 200ml, for high copy plasmids, 50ml do the job
Secondly, I read somewhere else that some people always add Chloramphenicol regardless the plasmid they were amplifying.
--> for low copy plasmids, you need a bigger amount of bacterias. That can cause a RNA contamination in your prep, due to diificulties to get rid of the amount coming from more bacterias. That's why people may add chloramphenicol, to get rid of transcription III. The bacterias stop growing and only replicates plasmids to be in general lines. That cause a redution of RNA quantity, without loosing benefits of a big culture
How much OD of bacteria do you achieve before starting the column procedure? (I believe that I don't achieve the plateau phase)
Hi do a 12-16h culture at 37°
Do you think there is any trouble if when defreezing your already transformed bacteria you put some of them directly in LB medium (with antibiotic) without using a plate.
I always try to plate. But i don't always plate (but in this case, it's for non-problem-causing plasmids)
Do you use Polypropile tubes to do MaxiPrep? Are there other type of tubes more convenient?
i don't have a clue regarding the composition of my tubes... But i think it's the case. They are falcon.
I usually grow for maxi prep in 250ml or 500ml. With 500ml, upto a 1mg of DNA.
With high copy plasmids, you can get more than 600ug from 100ml.
I never check OD. I have my miniprep culture in 4C and use 100uL to inoculate the maxi.
If your bacteria is not growing, start a 5ml culture in the morning from a single colony and then grow it for 9-12 hrs and then use it to inoculate the maxi.
I am not sure the material of the tubes. I guess it shouldnt matter much.
good luck !!!
I perform Maxi preps for transfection of HEK293E grown in suspension.
I usually achieve 850 micrograms to 1 mg per ml (eluting with a ml of TE). This is sufficient for transfecting a liter of HEK293E with polyethylenimine.
What kind of yields are you seeing?
-Matt
I have used the Qiagen maxipreps before and found that after precipitation and centrifugation, the pellet can be easily lost. Decanting is a bad idea, and even using a pipette tip can be a problem because the pellet breaks up into small peices due to shear suction. You have to be very careful and remove the supernatant with minimal disruption of the pellet. Alternatively, rather than do an isopropanol precipitation, perhaps do an ethanol precipitation. The pellet is easier to see and more compact.
I tend to precipitate with isopropanol and then place the solution into 15 eppendorf tubes. This makes it easier to see the pellet and thereby preventing its loss. I spin, wash and dry the pellets and then resuspend the pellet in water, transferring from tube to tube to concentrate the eluate.
I usually achieve 850 micrograms to 1 mg per ml (eluting with a ml of TE). This is sufficient for transfecting a liter of HEK293E with polyethylenimine.
What kind of yields are you seeing?
-Matt
Amazing! I though Maxiprep was up to 500ug, at least that's what is said in the handbook How much do I get? In my best attempt, only ~250ug (and I was happy with that until I've discovered how poor it is). I start to believe that I'm hopeless. :-(
Other thing I forgot to ask and it is vital to me to know is: pMEM is a low copy plasmid or high copy? I assumed it was a high copy, but I may be mistaken. I haven't been able to find this information in the net. Anyone knows?
About ML1975, I consider a really good approach, more time consuming but probably more likely to be successful.
About scolix, do you recommend me to try to grow my plasmid in more medium? I use only 100ml for 12-16h, as it has been correctly said by Fred. About "I have my miniprep culture in 4C and use 100uL to inoculate the maxi.", do you mean transformed bacteria from a miniprep? How many hour do you let them to grow before keeping at 4C? How long do you keep them at 4C?
One last thing. I've been doing the appropriate analytical gels and I do believe my problem is in the first part, just when you have your culture growth, then you add to the column and centrifuge (I collect my first sample from this supernatant). At that point my band in the gel is quite faint. I don't think it is the lysis step because with this new lysis blue reagent makes things even easier.
Sorry for so many questions. And thank you all very much.
I'm doing Maxi-Prep and I have achieved only one really successful. The rest of my attempts hasn't been really good, to be honest.
I wonder if anyone could answer the next questions and also give me some tips apart from those which are provided in the troubleshooting guide of Quiagen (I've tried some of them).
First of all, how much amount do you usually get when you do MaxiPrep (for 100ml of Lb, for ex)? (ug)
Secondly, I read somewhere else that some people always add Chloramphenicol regardless the plasmid they were amplifying.
How much OD of bacteria do you achieve before starting the column procedure? (I believe that I don't achieve the plateau phase)
Do you think there is any trouble if when defreezing your already transformed bacteria you put some of them directly in LB medium (with antibiotic) without using a plate.
Do you use Polypropile tubes to do MaxiPrep? Are there other type of tubes more convenient?
Please give me more suggestions.
Cheers
An apprentice
PS:
hi gsamsa,
first i agree with ML1975. sometimes the pellet is easy to lost so you have to be very carefully when you discard the SN. mark the pellet outside of the tube and observe when you decant the supernatant
another point: when i do my first preparation i have similar low amount of dna even though i used a high copy plasmid but after changing the competent cells my prep was very successful. maybe storage time of the c. cells is to long or (like in my case) try another bacterial strain for c.c.
good luck!
flausch
Dont worry, that u r getting low yields. I have to use 500ml of culture to get 1mg. So dont worry. If you need more DNA, then grow it in more medium. And also like I wrote before start a 5ml culture and grow during the day and then use the 5ml culture to inoculate the 500ml.
i dont know if pMEM is a low copy plasmid or high copy.
I grow minipreps for 14-16hrs. And I use most of it to check if the cloning went right. And store the rest of it at 4C. I would not use anything more than a week old in 4C. Better would be to retransform the DNA from miniprep (confirmed by restriction) and use a single colony to grow the maxi.
""One last thing. I've been doing the appropriate analytical gels and I do believe my problem is in the first part, just when you have your culture growth, then you add to the column and centrifuge (I collect my first sample from this supernatant). At that point my band in the gel is quite faint. I don't think it is the lysis step because with this new lysis blue reagent makes things even easier. ""
I dont understand you here. Please explain.
Don't worry about the last part, you have already given me an idea with that 500ml culture. It's one of the first things I will try.
I'm just starting to do all these cloning technique and I've never tried restriction digestion (Must be easy in most cases, I guess). I use instead PCR with specific primers, is this equally valid that restricion digestion? What is the main advantage of restricion digestion? To save time? Cost?
Anyone know about the pMEM, if it is low or high copy plasmid? Or where I could find such information?
I've tried this and it wasn't successful. How much bacteria pellet do you get? How much P1, P2 and P3 do you use then? Because when I tried with the double amount adviced for MaxiPrep I got a very viscous solution (P1 and P2) and I couldn't get rid of all the clumps.
In the protocol they advice you to centriufge twice the P1,P2 and P3 mixture and take an aliquot of the resultant supernatant containing the plasmid for running an analytical gel. I did this and I didn't get anything at all in my gel. So, I believe my problem is just before adding anything to the tip, when I get my pellet and I mix with P1, P2 and P3 (they containt the new lysis blue).
About the competent cells, both plasmid used the same batch of competent cells, the same day. The first one was all right (250ug) and the other one only 45ug.
First time I tried I got a good result (for me, because it was only 250ug). But with this plasmid, I'm very lost at the moment. Anyone could explain me what I'm doing wrong?
:-(
Have mercy on me, help me. Give me more advice and suggestions, please.