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pRSET purification - molecular biology (Feb/21/2011 )

Hi all ...

I'm trying to purify the pRSET vector; for this I am using XL1 competent cells, to transform them I put them in ice 30 minutes, then at 42 ║ C 90 sec and again on ice 2 minutes. After that, I incubated them at 37 ║ C for 1 hour and plated on LB plates with ampicillin.

By doing this I get several colonies, which re-planted in LB with ampicillin too, and let grow overnigth at 37 ║ C. to purification I'm using a commercial kit, but I can not get enough pure plasmid, only get 20-30ng/ul concentrations, that it not enough for any reaction. Wich may be the problem?? the kit?? cells?? plasmid??

Thank you very much, Greetings

Leo

-leo_cd-

leo_cd on Mon Feb 21 13:46:38 2011 said:


Hi all ...

I'm trying to purify the pRSET vector; for this I am using XL1 competent cells, to transform them I put them in ice 30 minutes, then at 42 ║ C 90 sec and again on ice 2 minutes. After that, I incubated them at 37 ║ C for 1 hour and plated on LB plates with ampicillin.

By doing this I get several colonies, which re-planted in LB with ampicillin too, and let grow overnigth at 37 ║ C. to purification I'm using a commercial kit, but I can not get enough pure plasmid, only get 20-30ng/ul concentrations, that it not enough for any reaction. Wich may be the problem?? the kit?? cells?? plasmid??

Thank you very much, Greetings

Leo


20-30 ng/ul shouldn't be too little, you should be able to work with that.

If it absolutely has to be higher, you can try:
* Growing in a 'better' media, i.e. Terrific Broth, in order to give you more cells for your miniprep
* Do a midi- or maxi-prep
* Grow more cells per o/n culture but spin them down in the same number of tubes (doubling the amount of cells input into each mini-prep)

-Kaioshin-

what overall yield you get after doing miniprep? ...if you have 700 ÁL of 30ng/ÁL its good ...if you have it in 20 ÁL it is probably low.

The pRSET has a pUC origin of replication and therefore is a high copy number plasmid that should give you high yields.
Maybe your low yield is due to the ampicillin and you have overgrown your cultur or inoculated with a too high cell density from your overnight culture.

Maybe you can describe in more detail what you did for growing your cells, what volume you used for the prep and in which volume you finally eluted?

Regards,
p

Kaioshin on Sat Mar 12 23:25:24 2011 said:


leo_cd on Mon Feb 21 13:46:38 2011 said:


Hi all ...

I'm trying to purify the pRSET vector; for this I am using XL1 competent cells, to transform them I put them in ice 30 minutes, then at 42 ║ C 90 sec and again on ice 2 minutes. After that, I incubated them at 37 ║ C for 1 hour and plated on LB plates with ampicillin.

By doing this I get several colonies, which re-planted in LB with ampicillin too, and let grow overnigth at 37 ║ C. to purification I'm using a commercial kit, but I can not get enough pure plasmid, only get 20-30ng/ul concentrations, that it not enough for any reaction. Wich may be the problem?? the kit?? cells?? plasmid??

Thank you very much, Greetings

Leo


20-30 ng/ul shouldn't be too little, you should be able to work with that.

If it absolutely has to be higher, you can try:
* Growing in a 'better' media, i.e. Terrific Broth, in order to give you more cells for your miniprep
* Do a midi- or maxi-prep
* Grow more cells per o/n culture but spin them down in the same number of tubes (doubling the amount of cells input into each mini-prep)

-pDNA-